Changes in isoagglutinin titers may have implications in the occurrence of hematological complications such as pure red cell aplasia or immune‐mediated hemolysis. Furthermore, isoagglutinin titers could reflect immunohematological reconstitution after transplantation. The objective of this study was to examine the relationship between donor‐derived isoagglutinins (DDIs) and graft‐versus‐host disease (GVHD). In total, 114 patients who underwent ABO‐incompatible allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed. Among these patients, 27.7% demonstrated increased donor‐derived isoagglutinins (IDDIs) against red blood cells (RBCs) of the recipient, and 32.8% of the patients showed DDIs that were not against RBCs of the recipient. Patients with acute GVHD and DDIs against RBCs of the recipient tended to have higher incidences of IDDIs that occurred before posttransplant day 60 compared with patients without acute GVHD (17.3 vs. 3.9%, P=0.058). In patients with acute GVHD, IDDIs occurred significantly earlier (mean, day 32 vs. 181, P=0.046), the period of elevation was shorter (mean, day 36 vs. 134, P=0.033), and the donors were younger (mean, 28 vs. 36 years, P=0.01) than those without GVHD. Moreover, significant correlations were found between IDDIs and acute GVHD. Taken together, these data underscore a possible role for humoral immunity in GVHD after HSCT. J. Clin. Lab. Anal. 22:383–390, 2008. © 2008 Wiley‐Liss, Inc.