Methylenetetrahydrofolate reductase is a pivotal enzyme in folate metabolism and 5-fluorouracil (5-FU) cytotoxicity. Two common single-nucleotide polymorphisms (SNPs), MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), reduce enzyme activity. Initially, these SNPs were claimed to predict clinical efficacy, but further studies have yielded contradictory results. We tested whether these two polymorphisms are determinants of clinical outcome in a large patient group with a long follow-up time. Patients and methods: We included 331 patients who had been treated with adjuvant 5-FU/leucovorin chemotherapy after intended curative resection between 1997 and 2003. Clinical data, including relapse rates, overall survival, and tumor stage, were collected. DNA was extracted from formalin-fixed tumor tissue and analyzed for the MTHFR 677C>T and 1298A>C SNPs with real-time PCR. Results: The MTHFR 677C>T and 1298A>C polymorphisms were not associated with survival or relapse-free survival (P > 0.2). The 677 CC genotype was associated to toxicity (odds ratio = 1.83, P = 0.01). Conclusions: The MTHFR 677C>T and 1298A>C polymorphisms probably do not predict efficacy of adjuvant 5-FU treatment in colorectal cancer after complete resection; however, the 677C>T polymorphism may be associated with lower toxicity in 5-FU treatment. Implementation of SNP analysis for these polymorphisms for individualized treatment is premature.