Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)-matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4(+) T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19(L)-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19(L)-specific CD4(+) T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8(+) mHag-specific T cells. They also lysed CD19(L)-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19(L)-derived HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.