BACKGROUND: Thiopurine S-methyltransferase (TPMT) genetic polymorphisms have been studied intensively in relation to thiopurine toxicity. In the present study, an improved pyrosequencing method was developed for TPMT genotyping and used to investigate the genotype frequency in Korean and Vietnamese populations. METHODS: Four-hundred Korean and 159 Vietnamese subjects were genotyped by pyrosequencing for TPMT 238G>C, 460G>A, 539A>T, and 719A>G variants, in order to determine the TPMT*2, *3A, *3B, *3C, and *6 alleles. RESULTS: TPMT*3C was found to be the most frequent allele in both the Korean (0.88%) and Vietnamese (2.83%) populations. TPMT*2, *3A, and *3B were not found in either of the study populations. Two Korean subjects were genotyped as TPMT*1/*6 (0.25%), while none of the Vietnamese had this genotype. The direct comparison of the frequencies of functional TPMT1/3C genotype showed significant difference between Korean and Vietnamese populations (chi2 test, p=0.0124). Duplex pyrosequencing methods for the detection of TPMT3C and TPMT6 were developed and validated to show 100% concordance with the results obtained by direct sequencing. CONCLUSIONS: The functional TPMT alleles in Korean and Vietnamese populations are TPMT*3C and TPMT*6. Duplex pyrosequencing for these alleles appears to be an accurate, rapid, and cost-effective method for genotyping TPMT in these Asian populations.