Thymidylate synthase (TYMS) converts dUMP to dTMP, the rate-limiting nucleotide in DNA synthesis. It is also the target for 5-flurouracil, the most common chemotherapy agent for treatment of colorectal cancer (CRC). We designed a case-control study to investigate the frequency of TS 1494del6 polymorphism in patients with CRC and controls from Mexican population. The study consists of 253 patients with CRC and 200 control subjects. Risk of CRC was estimated by use of Odds ratio (95% CI) analysis. The genotype 6bp-/6bp- in CRC patients was 18% (45/253) and in controls was 11% (22/200) with Odds ratio of 1.8 (1 - 4) with P = 0.059. When stratifying the training groups by age (< 50 years), male gender, stage III-IV and presence of metastasis, the genotype 6bp-/6bp- was as risk (p < 0.05). The genotype 6bp-/6bp- in the region untranslated 3' of TS gene, in the analyzed sample participates in important way in the development of CRC of the Mexican population.