Purpose: We analyzed the genotype and allele frequency of variable number tandem repeats (VNTR)-thymidylate synthase (TS) and its relationship with the disease evolution in colon cancer patients. Methods: We selected 24 paraffin-embedded colon cancer tissue samples from Mexican patients who received a 5-fluorouracil (5-FU)-based chemotherapy regimen. Tumor tissue was digested with proteinase K and genomic DNA was isolated by the standard method with phenol-chloroform extraction. Polymerase chain reaction (PCR) was performed for TS genotyping of VNTR and the results were evaluated directly in a stained agarose gel. Results: The allele frequency of 2 repeats (2R) was greater (0.66) than 3R (0.34) in metastatic colon cancer (x2=10.24; p=0.001); however, no difference in allelic distribution between 2R (0.54) and 3R (0.46) in non metastatic patients was observed (x2=0.640; p=0.424). Conclusion: Our results suggest that Mexican patients with colon cancer present differences in the allelic distribution, the 2R allele being the most frequent.