Sixty-six human enterovirus serotypes have been described using antibody neutralization, with antigenic variants defined within several serotypes. Despite the availability of sequence data for numerous enteroviruses, the molecular basis of serotype is unknown. Previous studies by others have identified four major phylogenetic groups within the human enteroviruses, but there has been no complete database of homologous sequences for all human enterovirus serotypes. We have determined the homologous partial VP2 sequences for the 12 prototype strains for which VP2 sequence was unavailable and for eight well-characterized antigenic variants. Phylogenetic analysis of all prototype strains produced four major groups, consistent with published enterovirus phylogenies. Many antigenic variants, however, failed to cluster with their respective prototype strains, suggesting that this portion of VP2 may be inappropriate for consistent molecular inference of serotype and for detailed study of enterovirus evolution.