The role of thymidylate synthase (TS) is essential as a key rate-limiting enzyme in DNA synthesis. It is the primary target of fluorouracil and its derivates in colorectal cancer. In this study, TS mRNA expression was examined in primary tumor and normal tissues from 76 patients with high- risk stage II/III colorectal cancer by laser capture microdissection and polymerase chain reaction. Thirty (39.47%) patients were found to have higher TS expression in primary tumors with earlier stage (P = 0.018), lower histological grades (P = 0.001) and high frequency microsatellite instability (P = 0.000). Multivariate analysis showed that microsatellite instability, histological grade and number of lymph nodes examined are independent prognostic markers.