The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1-H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238 and 484. Haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while 80-90% of the white population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 HA subjects (188 black, 206 white), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most HA subjects, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, and peptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, the BDD-FVIII proteins were markedly more reactive than the full-length FVIII products with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal may expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.