A new isolate of hepatitis A virus (HAV), CY-145, was isolated from stool specimens obtained from cynomolgus macaques naturally infected with this agent. Sequence analysis of the capsid region of the genome indicated that this virus differed from other sequenced HAV strains by about 20% at the nucleotide level and 7% at the amino acid level. Two amino acid residues (residues 70 of VP3 and 102 of VP1), previously identified as constituting an immunodominant site and conserved in all sequenced HAVs, were changed in the CY-145 virus. Sequence analysis of a second cynomolgus HAV isolate (CY-55), which came from a different geographical location, showed the same amino acid replacement at these two sites. In addition both isolates had an amino acid substitution at the VP3-VP1 cleavage site. These data suggest that the cynomolgus HAV differs genetically and antigenically from all other sequenced HAVs.