Human leukocyte antigen (HLA) high-resolution matching is routinely performed in the case of hematopoietic stem cell transplantation. Misdiagnosis of rare or new variants will result in additional unknown mismatches that can trigger graft versus host disease and/or graft failure. Using a mono allelic sequencing strategy, we obtained >90% first-step sequence-based high-resolution typing results, conforming to the standards set down by the European Federation for Immunogenetics. During this routine analysis, eight new HLA alleles were identified, including one DRB1*12 null variant, DRB1*12:24N and three other coding mutants, resulting in a change of properties of the corresponding antigens.