Isolates of hepatitis A virus (HAV) are of a single serotype, with human isolates being categorised within four genotypes. In addition, there are three genotypes exclusively associated with Old World monkeys. In some geographical regions, related isolates cluster suggesting endemic spread of the virus, while in other regions several genotypes circulate. Virtually no data are available with regard to the genetic relatedness of South African (SA) strains of HAV. A 177 base segment within the VP1 region and a 168 base segment encompassing the putative VP1/P2A junction of 20 clinical and one environmental wild-type isolate(s) of HAV from SA were amplified by reverse transcriptase-polymerase chain reaction. The nucleotide sequences from the SA isolates showed > 85% nucleic acid sequence identity with published sequences for HAV strains from genotype I, with the majority of strains (81%) clustering within subgenotype IB and the remainder in subgenotype IA. A high degree of conservation was noted between the predicted amino acid sequences from SA clinical isolates and isolates from the rest of the world. Data presented indicate that in SA there is a circulating population of endemic HAVs from two distinct subgenotypes. This study provides valuable new data on the genetic relatedness of HAVs from southern Africa and the distribution of subgenotype IB.