The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.