Minor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic T-lymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity.