Human minor histocompatibility antigens (mHag) are target antigens of the graft‐versus‐leukemia response observed after allogeneic HLA‐identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage‐specific mHag HB‐1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA‐B44. The HB‐1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR‐RFLP assay to perform HB‐1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA‐B44‐matched sibling pairs for HB‐1H is 2.8%, whereas recipient disparity for HB‐1Y is expected to be 12.4%. Therefore, we addressed whether the HB‐1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA‐B44 molecules and that the H/Y substitution has noinfluence on formation of epitope precursor peptides by 20 S proteasome‐mediated degradation. More directly, CTL recognizing the naturally presented HB‐1Y peptide could be generated from a HB‐1H homozygous donor using peptide‐pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB‐1 allelic homologues. HB‐1 is the first human mHag described that induces bi‐directional allogeneic CTL responses that may contribute to a specific graft‐versus‐leukemiaresponse following allogeneic stem cell transplantation.