HLA-A*31:01 has been associated with carbamazepine-induced hypersensitivity reactions. HLA-A*31:01 genetic testing is recommended before the initiation of carbamazepine therapy. A novel real-time PCR assay was designed for HLA-A*31:01 detection by allele-specific primers and TaqMan minor groove binding probes. The genotyping results in 100 subjects by the established method who were in 100% agreement with the sequencing-based typing method. The assay presents a sensitivity of 1 (95% CI: 0.69-1.00), a specificity of 1 (95% CI: 0.96-1.00) and a positive and negative predictive value of 1. The carrier rates of HLA-A*31:01 in Tibetan (n=45), Han Chinese (n=100), Miaos (n=48) and Khalkhas (n=48) were 22.2, 10, 4.2 and 18.8%, respectively. This assay is reliable to detect HLA-A*31:01 and would be useful to prevent carbamazepine-induced hypersensitivity reactions.