Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.