Minor histocompatibility antigens (mHAs) are known targets of donor T cells after allogeneic hematopoietic stem cell transplantation (HSCT). In contrast, B-cell responses to mHAs have not been extensively characterized and the clinical significance of antibodies to mHAs is unknown. We tested 121 patients who underwent HSCT and 134 healthy donors for immunoglobulin G (IgG) antibodies against 5 mHAs encoded by genes on the Y chromosome (DBY, UTY, ZFY, RPS4Y, and EIF1AY). Antibodies to at least one H-Y protein developed in 52% of male patients with female donors compared with 8.7% of male patients with male donors (P < .0001), and in 41.4% of healthy females compared with 7.8% of healthy males (P < .0001). H-Y antibodies develop 4 to 12 months after transplantation and persist for long periods. The clinical significance of H-Y antibodies was characterized in 75 male patients with hematologic malignancies who received stem cells from female donors (F → M HSCT). The presence of H-Y antibodies correlated with chronic graft-versus-host disease (GVHD) by univariate (odds ratio [OR] = 15.5; P < .0001) and multivariable logistic regression analysis (OR = 56.5; P < .0001). Antibody response to Y-chromosome encoded histocompatibility antigens (H-Y antigens) was also associated with maintenance of disease remission (P < .0001). B cells may provide a new target for immune intervention in chronic GVHD.