In allogenic hematopoietic stem-cell transplantation, an effect of HLA locus mismatch in allele level on clinical outcome has been clarified. However, the effect of each HLA allele mismatch combination is little known, and its molecular mechanism to induce acute graft-versus-host disease (aGVHD) remains to be elucidated. A total of 5210 donor-patient pairs who underwent transplantation through Japan Marrow Donor Program were analyzed. All HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 alleles were retrospectively typed in all pairs. The impacts of the HLA allele mismatch combinations and amino acid substitution positions in 6 HLA loci on severe aGVHD were analyzed. A total of 15 significant high-risk HLA allele mismatch combinations and 1 HLA-DRB1-DQB1 linked mismatch combinations (high-risk mismatch) for severe aGVHD were identified, and the number of high-risk mismatches was highly associated with the occurrence of severe aGVHD regardless of the presence of mismatch combinations other than high-risk mismatch. Furthermore, 6 specific amino acid substitution positions in HLA class I were identified as those responsible for severe aGVHD. These findings provide evidence to elucidate the mechanism of aGVHD on the basis of HLA molecule. Furthermore, the identification of high-risk mismatch, that is, nonpermissive mismatch, would be beneficial for the selection of a suitable donor.