Genetic relationships of 50 SAT-1 type foot-and-mouth disease viruses were determined by phylogenetic analysis of an homologous 417 nucleotide region encoding the C-terminal half of the VP1 gene and part of the 2A segment. Viruses obtained from persistently-infected African buffalo populations were selected in order to assess the regional genetic variation within the host species and compared with ten viruses recovered from recent and historical cases of clinical infection. Phylogenetic reconstructions identified three independently evolving buffalo virus lineages within southern Africa, that correspond with the following discrete geographic localities: (1) South Africa and southern Zimbabwe, (2) Namibia, Botswana and western Zimbabwe, and (3) Zambia, Malawi and northern Zimbabwe. This strict geographic grouping of viruses derived from buffalo was shown to be useful for determining the origin of recent SAT-1 epizootics in livestock. The percentage of conserved amino acid sites across the 50 SAT-1 viruses compared in this study was 50%. Most mutations were clustered within three discrete hypervariable regions, which coincide with the immunogenic G-H loop, H-1 loop and C-terminus region of the protein. Despite the high levels of variation within the primary sequence, secondary structural features appear to be conserved.