Epstein-Barr virus (EBV) infects B cells and ~95% of adults are infected. EBV glycoprotein gp42 is essential for entry of virus into B cells. EBV gp42 binds to the β1 chain of HLA-DQ, -DR, and -DP on B cells, and uses these molecules for infection. To investigate if certain HLA-DQ alleles are associated with EBV seronegativity, we recruited ~3,300 healthy adult blood donors, identified 106 EBV-seronegative individuals, and randomly selected a control group of EBV-seropositive donors from the donor pool. A larger than expected proportion of EBV-seronegative subjects were HLA-DQ β1 *04/*05 and *06/*06, and to a lesser extent, *02/*03, compared with the control group, while a larger than expected portion of EBV-seropositive persons were HLA-DQ β1 *02/*02. We examined the ability of EBV gp42 to bind to different HLA-DQ molecules using human and mouse cells stably expressing these alleles. EBV gp42 bound less effectively to cells expressing HLA-DQ β1 *04/*05, *06/*06, or *03/*03 than to cells expressing HLA-DQ β1 *02/*02. These data are consistent with our observations of increased EBV seronegativity with DQ β1 *04/*05 or *06/*06 alleles. These findings emphasize the importance of a single genetic locus (HLA-DQ β1) to influence infectivity with EBV.