Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs that are commonly used to treat a wide range of conditions. It is now well established that interpatient variation in sensitivity to these drugs is due to point mutations in the TPMT gene. The mutant alleles TPMT*2 (238G>C), TPMT*3A (460G>A, 719A>G), TPMT*3B (460G>A), and TPMT*3C (719A>G) account for 80-95% of TPMT deficiency observed in Caucasian populations. In this paper, we describe a novel, multiplex, allele-specific polymerase chain reaction (PCR) method that detects the 238G>C, 460G>A, and 719A>G mutations, allowing for the simultaneous identification of TPMT*2 and TPMT*3 alleles. The assay is internally controlled, robust, and does not require the use of restriction endonucleases. Therefore, the assay is not prone to erroneous readings due to incomplete restriction digestion, as documented for existing PCR restriction fragment length polymorphism (RFLP) assays of TPMT.