BACKGROUND: The Diego blood group presents a major polymorphic site at Residue 854, causing a proline (Di(b) antigen) to leucine (Di(a) antigen) substitution. Di(a) alloimmunization has been observed among Asian and Native South American populations. Considering that Brazilians represent a genetically diverse population, and considering that we have observed a high incidence of Di(a) alloimmunization, we typed HLA-DRB1 alleles in these patients and performed in silico studies to investigate the possible associated mechanisms. STUDY DESIGN AND METHODS: We studied 212 alloimmunized patients, of whom 24 presented immunoglobulin G anti-Di(a) , 15 received Di(a+) red blood cells and were not immunized, and 1008 were healthy donors. HLA typing was performed using commercial kits. In silico analyses were performed using the TEPITOPEpan software to identify Diego-derived anchor peptide binding to HLA-DRB1 molecules. Residue alignment was performed using the IMGT/HLA for amino acid identity and homology analyses. RESULTS: HLA-DRB1*07:01 allele was overrepresented in Di(a) -alloimmunized patients compared to nonimmunized patients and to healthy donors. Two motifs were predicted to be potential epitopes for Di(a) alloimmunization, the WVVKSTLAS motif was predicted to bind several HLA-DR molecules, and the FVLILTVPL motif exhibited highest affinity for the HLA-DRB1*07:01 molecule. Pocket 4 of the DRB1*07:01 molecule contained specific residues not found in other HLA-DRB1 molecules, particularly those at Positions 13(Y), 74(Q), and 78(V). CONCLUSION: Individuals carrying the HLA-DRB1*07:01 allele present an increased risk for Di(a) alloimmunization. The identification of susceptible individuals and the knowledge of potential sensitization peptides are relevant approaches for transfusion care, diagnostic purposes, and desensitization therapies.