The important cellular proteins for 5-fluorouracil (5-FU) metabolism are the major target enzymes, thymidylate synthase, and the rate-limiting enzyme in the degradation pathway, dihydropyrimidine dehydrogenase. Adverse drug reactions to 5-FU-based chemotherapy have been reported to be in part the result of polymorphisms in the thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) genes. Therefore, we examined the type and frequency of polymorphisms in the TYMS and DPYD genes in 100 healthy Korean individuals and compared these findings with 21 patients with colorectal cancer who had a grade 3 or greater toxic response to 5-FU treatment. Genotyping analysis of the promotor enhancer region (TSER) and the 3'-untranslated region (3'-UTR) of the TYMS gene as well as haplotype analysis were conducted in all 121 study participants. For the TSER and the 3'-UTR of the TYMS gene, similar genotypes and allele frequencies were observed in control subjects and patients. For the haplotype analysis of the single nucleotide polymorphism G > C at the 12th nucleotide of the second repeat of the 3R allele of the TSER, different haplotype frequencies were noted in comparisons between the two groups; in addition, we found that the 3RC-del 6bp was significantly associated with severe toxicity with 5-FU treatment. Extensive polymorphisms in the DPYD gene were observed; in addition, four polymorphisms were related to the known DPYD allelic variants or to allelic variants that alter protein structure, among which the most common polymorphism was 1627A > G, observed in 20.5% of all alleles. The 496A > G allele and a novel 1774C > T allele were identified in two patients. The DPYD*2A allele, causing exon 14 skipping, was not identified in the study group. The findings, from Korean patients with colon cancer, suggest that polymorphisms of the DPYD gene are not associated with an increased risk for toxic response to 5-FU. These findings suggest that there may be an important relationship between the TYMS haplotypes examined and 5-FU toxicity. The novel variant in the DPYD gene, identified in this study, should be further investigated to confirm its functional significance. A large sample is required before DPYD or TYMS genotyping could be used as markers for individualized treatment of patients with colorectal cancer.