The clinical definition of smallpox is an illness with acute onset of fever equal to or greater than followed by a rash characterized by firm, deep seated vesicles or pustules in the same stage of development without other apparent cause. If a clinical case is observed, smallpox is confirmed using laboratory tests.

Microscopically, poxviruses produce characteristic cytoplasmic inclusions, the most important of which are known as Guarnieri bodies, and are the sites of viral replication. Guarnieri bodies are readily identified in skin biopsies stained with hematoxylin and eosin, and appear as pink blobs. They are found in virtually all poxvirus infections but the absence of Guarnieri bodies cannot be used to rule out smallpox. The diagnosis of an orthopoxvirus infection can also be made rapidly by electron microscopic examination of pustular fluid or scabs. All orthopoxviruses exhibit identical brick-shaped virions by electron microscopy. If particles with the characteristic morphology of herpesviruses are seen this will eliminate smallpox and other orthopoxvirus infections.

Definitive laboratory identification of variola virus involves growing the virus on chorioallantoic membrane (part of a chicken embryo) and examining the resulting pock lesions under defined temperature conditions. Strains may be characterized by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Serologic tests and enzyme linked immunosorbent assays (ELISA), which measure variola virus-specific immunoglobulin and antigen have also been developed to assist in the diagnosis of infection.

Chickenpox was commonly confused with smallpox in the immediate post-eradication era. Chickenpox and smallpox can be distinguished by several methods. Unlike smallpox, chickenpox does not usually affect the palms and soles. Additionally, chickenpox pustules are of varying size due to variations in the timing of pustule eruption: smallpox pustules are all very nearly the same size since the viral effect progresses more uniformly. A variety of laboratory methods are available for detecting chickenpox in evaluation of suspected smallpox cases.

The earliest procedure used to prevent smallpox was inoculation (known as variolation after the introduction of smallpox vaccine to avoid possible confusion), which likely occurred in India, Africa, and China well before the practice arrived in Europe. The idea that inoculation originated in India has been challenged, as few of the ancient Sanskrit medical texts described the process of inoculation. Accounts of inoculation against smallpox in China can be found as early as the late 10th century, and the procedure was widely practiced by the 16th century, during the Ming dynasty. If successful, inoculation produced lasting immunity to smallpox. Because the person was infected with variola virus, a severe infection could result, and the person could transmit smallpox to others. Variolation had a 0.5–2 percent mortality rate, considerably less than the 20–30 percent mortality rate of the disease. Two reports on the Chinese practice of inoculation were received by the Royal Society in London in 1700; one by Dr. Martin Lister who received a report by an employee of the East India Company stationed in China and another by Clopton Havers.

Lady Mary Wortley Montagu observed smallpox inoculation during her stay in the Ottoman Empire, writing detailed accounts of the practice in her letters, and enthusiastically promoted the procedure in England upon her return in 1718. In 1721, Cotton Mather and colleagues provoked controversy in Boston by inoculating hundreds. In 1796, Edward Jenner, a doctor in Berkeley, Gloucestershire, rural England, discovered that immunity to smallpox could be produced by inoculating a person with material from a cowpox lesion. Cowpox is a poxvirus in the same family as variola. Jenner called the material used for inoculation vaccine, from the root word "vacca", which is Latin for cow. The procedure was much safer than variolation, and did not involve a risk of smallpox transmission. Vaccination to prevent smallpox was soon practiced all over the world. During the 19th century, the cowpox virus used for smallpox vaccination was replaced by vaccinia virus. Vaccinia is in the same family as cowpox and variola, but is genetically distinct from both. The origin of vaccinia virus and how it came to be in the vaccine are not known. According to Voltaire (1742), the Turks derived their use of inoculation to neighbouring Circassia. Voltaire does not speculate on where the Circassians derived their technique from, though he reports that the Chinese have practiced it "these hundred years".

The current formulation of smallpox vaccine is a live virus preparation of infectious vaccinia virus. The vaccine is given using a bifurcated (two-pronged) needle that is dipped into the vaccine solution. The needle is used to prick the skin (usually the upper arm) a number of times in a few seconds. If successful, a red and itchy bump develops at the vaccine site in three or four days. In the first week, the bump becomes a large blister (called a "Jennerian vesicle") which fills with pus, and begins to drain. During the second week, the blister begins to dry up and a scab forms. The scab falls off in the third week, leaving a small scar.

The antibodies induced by vaccinia vaccine are cross-protective for other orthopoxviruses, such as monkeypox, cowpox, and variola (smallpox) viruses. Neutralizing antibodies are detectable 10 days after first-time vaccination, and seven days after revaccination. Historically, the vaccine has been effective in preventing smallpox infection in 95 percent of those vaccinated. Smallpox vaccination provides a high level of immunity for three to five years and decreasing immunity thereafter. If a person is vaccinated again later, immunity lasts even longer. Studies of smallpox cases in Europe in the 1950s and 1960s demonstrated that the fatality rate among persons vaccinated less than 10 years before exposure was 1.3 percent; it was 7 percent among those vaccinated 11 to 20 years prior, and 11 percent among those vaccinated 20 or more years prior to infection. By contrast, 52 percent of unvaccinated persons died.

There are side effects and risks associated with the smallpox vaccine. In the past, about 1 out of 1,000 people vaccinated for the first time experienced serious, but non-life-threatening, reactions, including toxic or allergic reaction at the site of the vaccination (erythema multiforme), spread of the vaccinia virus to other parts of the body, and to other individuals. Potentially life-threatening reactions occurred in 14 to 500 people out of every 1 million people vaccinated for the first time. Based on past experience, it is estimated that 1 or 2 people in 1 million (0.000198 percent) who receive the vaccine may die as a result, most often the result of postvaccinial encephalitis or severe necrosis in the area of vaccination (called progressive vaccinia).

Given these risks, as smallpox became effectively eradicated and the number of naturally occurring cases fell below the number of vaccine-induced illnesses and deaths, routine childhood vaccination was discontinued in the United States in 1972, and was abandoned in most European countries in the early 1970s. Routine vaccination of health care workers was discontinued in the U.S. in 1976, and among military recruits in 1990 (although military personnel deploying to the Middle East and Korea still receive the vaccination). By 1986, routine vaccination had ceased in all countries. It is now primarily recommended for laboratory workers at risk for occupational exposure.

Alastrim, also known as variola minor, was the milder strain of the variola virus that caused smallpox. The last known case of variola minor was in Somalia, Africa in 1977. Smallpox was formally declared eradicated in May 1980.

Variola minor is of the genus orthopoxvirus, which are DNA viruses that replicate in the cytoplasm of the affected cell, rather than in its nucleus. Like variola major, alastrim was spread through inhalation of the virus in the air, which could occur through face-to-face contact or through fomites. Infection with variola minor conferred immunity against the more dangerous variola major.

Variola minor was a less common form of the virus, and much less deadly. Although alastrim had the same incubation period and pathogenetic stages as smallpox, alastrim is believed to have had a mortality rate of less than 1%, as compared to smallpox's 30%.

Because alastrim was a less debilitating disease than smallpox, patients were more frequently ambulant and thus able to infect others more rapidly. As such, variola minor swept through the USA, Great Britain, and South Africa in the early 20th century, becoming the dominant form of the disease in those areas and thus rapidly decreasing mortality rates.

Alastrim was also called white pox, kaffir pox, Cuban itch, West Indian pox, milk pox, and pseudovariola.

Like smallpox, alastrim has now been totally eradicated from the globe thanks to the 1960s Global Smallpox Eradication campaign. The last case of indigenous variola minor was reported in a Somalian cook, Ali Maow Maalin, in October 1977, and smallpox was officially declared eradicated worldwide in May 1980.