Many autoimmune diseases share a genetic association with the presence or absence of human leukocyte antigen (HLA)-DQB1*0602, including type I diabetes, multiple sclerosis, and narcolepsy. High-resolution HLA typing to determine the presence of this allele is cumbersome and expensive by currently available techniques. We present a real-time polymerase chain reaction (PCR) assay for the identification of HLA-DQB1*0602, using sequence-specific primers and probes, that provides rapid and sensitive identification of this allele, involves minimal hands-on time, and provides major cost savings compared with existing methods. The assay allows the simultaneous determination of both the presence and the number of copies of this allele. Because there is no post-PCR handling, the risk of contamination is avoided. We have validated the assay using 44 blinded and 32 unblinded samples, previously typed by standard techniques, which were identified with 100% accuracy, sensitivity, and specificity. Furthermore, using a narcolepsy cohort of 722 subjects, we demonstrated the robustness of the assay to analyze DNA isolated from buccal swabs, demonstrating the applicability of this assay as a suitable approach for population-based studies.