Thymidylate synthase (TS) is a target enzyme of 5-fluorouracil (5-FU). TS has a polymorphic repeated sequence in the 5'-UTR and the polymorphism is associated with TS protein expression. TS polymorphism has been reported to link with the efficacy of 5-FU-based chemotherapy in colorectal cancer. In this study, we examined whether the association among TS polymorphism, TS mRNA expression and TS protein expression is also observed in gastric cancer tissues and whether the TS polymorphism is a prognostic factor for patients with gastric cancer. We quantified TS mRNA isolated from 115 gastric cancer tissues by real-time reverse transcription PCR and TS protein in 72 available samples by the fluoro-dUMP binding assay. These values were compared with TS genotypes of the samples determined by a PCR assay. The tumor with the 3R/3R genotype had higher TS protein expression than that with the 2R/3R genotype, although there is no association between the TS genotype and mRNA expression. The result suggests that mRNA translation is responsible for the genotype-dependent difference of TS protein expression, as is consistent with our previous observation in colorectal cancer. This association encouraged us to further examine the link of the TS genotype with the stage of disease and the prognosis of the patients. The clinicopathological analysis showed that gastric cancers with the 3R/3R genotype are at a more advanced stage than those with the combined 2R/2R and 2R/3R genotype. We observed a longer survival in those patients with the combined 2R/2R and 2R/3R genotype, compared with the 3R/3R genotype, although it did not reach significance when the patients who had received the oral fluoropyrimidines therapy were analyzed. These results warrant further large-scale clinical study of the role of the TS genotyping for the prediction of efficacy using 5-FU-based chemotherapy and prognosis in gastric cancer.