The reported outcome of hematopoietic stem cell transplantation (HSCT) from HLA-mismatched family members has been inconsistent. The object of this study was to evaluate the true impact of HLA-mismatch by using recent data from a homogenous population, excluding HSCT procedures that used graft manipulations, and by considering genotypic matching. Clinical data of 2947 patients who underwent allogeneic HSCT for leukemia or myelodysplastic syndrome were extracted from the database of the Japan Society for Hematopoietic Cell Transplantation. The main outcome measures were survival and the incidence of graft-versus-host disease (GVHD). The presence of serologic HLA-mismatch, higher age, and high-risk disease were identified as independent risk factors for both shorter survival and the development of grade III to IV acute GVHD. The impact of HLA-mismatch on survival was more relevant in standard-risk patients. These findings persisted when we used genotypic HLA matching. Survival after one-locus-mismatched HSCT was equivalent to that after HLA-matched unrelated HSCT. We concluded that when a one-locus-mismatched family donor is available for high-risk patients, immediate HSCT using this donor is warranted. In standard-risk patients, however, survival after one-locus-mismatched HSCT is significantly shorter than that after HLA-matched HSCT, and the indications for HSCT should be considered carefully.