Bone marrow transplantation from unrelated donors (UR-BMT) has been considered to be effective for patients with hematological malignancies who have no suitable related donor. However, disparities of HLA between a recipient and a donor increase the risk of severe acute graft-versus-host disease (GVHD). We evaluated GVHD prophylaxis using tacrolimus and methotrexate for HLA-A, B, or DRB1 genotypically mismatched UR-BMT. Fifty-five patients were enrolled in this study. The incidence of grade III to IV acute GVHD was 23.6% for all patients. No significant difference in the incidence of grade III to IV acute GVHD was observed between HLA-A or B 1 locus mismatch transplantation (18.8%) and HLA-DRB1 1 locus mismatch transplantation (16.7%) (P = 0.96). The incidence of chronic GVHD was 71.7%. Disease-free survival at 5 years was 53.2% for patients with standard risk disease and 24.5% for patients with high-risk disease. Patients with chronic GVHD exhibited better disease-free survival than those without chronic GVHD (53.2 vs. 30.9%, P = 0.011). Twenty patients (36.4%) had a relapse of leukemia and 14 of them died of recurrent leukemia. This study indicates tacrolimus and methotrexate can lower the risk of severe acute GVHD after HLA-A, B, or DRB1 genotypically 1 locus mismatched UR-BMT.