The variable number of tandem repeat (VNTR) of thymidylate synthase (TS) gene, mainly 2 repeat (2R) and 3 repeat (3R), is one of the genetic variations that can potentially predict the effectiveness of 5-fluorouracil-based chemotherapy. In this study we identified an additional single nucleotide polymorphism (SNP) in the VNTR of TS, followed by functional and clinical analysis of the SNP. Two-hundred fifty eight tumor samples were obtained from patients with primary colorectal adenocarcinoma. We observed three different patterns of electrophoresis by analysis of the VNTR with 2R/3R heterozygote. The sequencing results revealed a SNP, G/C polymorphism, within the 28-bp repeat component of TS VNTR. Each polymorphic allele was assigned as 2G, 2C, 3G, or 3C according to the combination of SNP and VNTR. Functional analysis showed that the plasmid construct with 3G sequence had three to four times greater efficiency of translation than other polymorphic sequences. 3R allele in colorectal cancer was subdivided into around half by the SNP, indicating its commonness among Japanese. TS genotypes of the patients with colorectal cancer were classified into high expression type (2R/3G, 3C/3G, and 3G/3G) and low expression type (2R/2R, 2R/3C, and 3C/3C). The patients who received oral fluoropyrimedines survived longer than the patients with no treatment in the group of low expression type. No benefit of oral fluoropyrimedines was observed in the group of high expression type. These results suggest that the double polymorphism in the TS tandem repeat sequence, the SNP and the VNTR, may provide a potential for more effective prediction of the clinical outcome of 5-fluorouracil-based chemotherapy.