Thymidylate synthase (TS) is a key enzyme in folate metabolism, a pathway that is important in colorectal carcinogenesis. We investigated the role of functional polymorphisms in the TS 5′-UTR promoter enhancer region (TSER, 3 or 2 repeats of a 28-bp sequence) and the 3′-UTR (1494delTTAAAG) and their association with colon tumor characteristics, including tumor stage and acquired mutations in p53, Ki-ras and microsatellite instability. Data from a population-based incident case-control colon cancer study in northern California, Utah and Minnesota (1,206 cases, 1,962 controls) was analyzed using unordered polytomous logistic regression models. In both men and women, individuals with variant TS alleles were at reduced risk of having an advanced stage tumor (metastatic disease: OR = 0.35, 95% CI: 0.2-0.6 vs. wildtype TSER and 3′-UTR). Stage-adjusted survival did not differ by genotype. Men with 1 or 2 variant alleles in both the TSER and 3′-UTR genotypes had a 50% reduced risk of a p53-positive tumor (OR = 0.5, 95% CI: 0.3-0.9 vs. homozygous wildtype TSER and 3′-UTR). Women with 1 or 2 variant alleles for either the TSER or 3′-UTR polymorphism had reduced risk of having any colon tumor that did not vary by mutation status. This study provides some support for associations between TS genotype and colon cancer tumor characteristics.