We retrospectively analyzed the impact of HLA mismatching (HLA-A, -B, -C, -DRB1, -DQB1) using molecular typing on transplant outcome for 301 patients with acquired severe aplastic anemia (SAA) who received unrelated bone marrow transplantation through the Japan Marrow Donor Program. Additional impact of HLA-DPB1 mismatching was analyzed for 10/10 or 9/10 HLA allele matched pairs (n = 169). Of the 301 recipient-donor pairs, 101 pairs (33.6%) were completely matched at 10/10 alleles, 69 pairs (23%) were mismatched at one allele, and 131 (43.5%) were mismatched at two or more alleles. Subjects were classified into 5 subgroups: complete match group (group I), single allele mismatch group (groups II and III), multiple alleles restricted to HLA-C, -DRB1, and -DQB1 mismatch group (group IV), and others (group V). Multivariate analysis indicated that only HLA disparity of group V was a significant risk factor for poor survival and grade II-IV acute graft-versus-host disease. HLA-DPB1 mismatching was not associated with any clinical outcome. We recommend the use of an HLA 10/10 allele-matched unrelated donor. However, if such a donor is not available, any single allele mismatched donor or multiple allele (HLA-C, -DRB1, -DQB1) mismatched donors are acceptable as an unrelated donor for SAA patients.