In transplantation, development of humoral alloimmunity against donor HLA is a major cause of organ transplant failure, but our ability to assess the immunological risk associated with a potential donor-recipient HLA combination is limited. We hypothesized that the capacity of donor HLA to induce a specific alloantibody response depends on their structural and physicochemical dissimilarity compared with recipient HLA. To test this hypothesis, we first developed a novel computational scoring system that enables quantitative assessment of surface electrostatic potential differences between donor and recipient HLA molecules at the tertiary structure level [three-dimensional electrostatic mismatch score (EMS-3D)]. We then examined humoral alloimmune responses in healthy females subjected to a standardized injection of donor lymphocytes from their male partner. This analysis showed a strong association between the EMS-3D of donor HLA and donor-specific alloantibody development; this relationship was strongest for HLA-DQ alloantigens. In the clinical transplantation setting, the immunogenic potential of HLA-DRB1 and -DQ mismatches expressed on donor kidneys, as assessed by their EMS-3D, was an independent predictor of development of donor-specific alloantibody after graft failure. Collectively, these findings demonstrate the translational potential of our approach to improve immunological risk assessment and to decrease the burden of humoral alloimmunity in organ transplantation.