BACKGROUND Thiopurine S-methyltransferase (TPMT) is involved in the toxicity and therapeutic efficacy of thiopurine drugs, and its gene exhibits genetic polymorphisms that differ across diverse populations. Four TPMT polymorphisms (TPMT*2, *3A, *3B and *3C) account for 80-95% of alleles that cause reduced enzyme activity. To date, only a single study in the Mexican population involving 108 individuals has been performed, but the regional and ethnic origin of this population was not described. Accordingly, information about the TPMT polymorphism in the Mexican population is limited. OBJECTIVE To determine the TPMT allele and genotype frequencies in a sample of newborns from Mexico City. METHODS Three hundred and sixty DNA samples from unrelated, anonymous individuals were obtained from dried blood spots collected on filter paper as part of the Newborn Screening National Program. Allele-specific polymerase chain reaction for the TPMT*2 allele and PCR restriction fragment length polymorphism for TPMT*3A, TPMT*3B, TPMT*3C alleles were used to determine the respective allelic and genotypic frequencies. CONCLUSIONS Of 720 TPMT alleles analysed, 49 (6.81%) were deficiency alleles. The most common deficiency allele was TPMT*3A (5.69%), followed by TPMT*3C (0.56%), TPMT*3B (0.28%) and TPMT*2 (0.28%). Fourty-five newborns were heterozygous for one mutant allele (12.5%) and two showed a genotype with two deficiency alleles (0.56%). Despite its unique ethnic composition, our Mexican population exhibited variant allele frequencies that were similar to some Caucasian populations. CONCLUSIONS Our data suggest that approximately 1 in 180 persons born in Mexico City might have low or undetectable TPMT enzyme activity, a frequency that, overall, is somewhat higher than that reported for Caucasian populations generally (1 in 300).