Although its cause is poorly understood, situs ambiguous has been linked to family history of malformations and maternal cocaine use, suggesting both genetic and environmental factors play a role. Several genes in the TGF-beta pathway, which controls left-right patterning of viseral organs across the body axis, have been indicated in sporadic and familial cases of atrial isomerism.

There does not appear to be a screening method for prevention of heterotaxy syndrome. However, genetic testing in family members that display atrial isomerism or other cardiac malformations may help to discern risk for additional family members, especially in X-linked causes of heterotaxy syndrome.

Several genes have been identified in normal development of the right-left axis. These genes have been extensively researched. Gene mutations that lead to atrial isomerism is a growing area of research. Mutations in genes that encode proteins in the TGF-beta pathway, including NODAL, NKX2-5, and ZIC3, have been linked to tetralogy of fallot and hypoplastic left heart syndrome. Mutations in the ZIC3 gene, which encodes for the first zinc finger transcription factor, is linked to a 50% risk of atrial isomerism in families. It is also an X-linked disorder, so testing for ZIC3 mutations is highly encouraged in male births.

The most prevalent and best characterized genetic associations of heterotaxy include: