Microdissection is a reliable technique and is extensively used in many cancer studies. We sought to verify the importance of the microdissection technique in molecular analysis of irradiated rectal cancer specimens. Forty patients with rectal cancer underwent 5-fluorouracil based chemoradiotherapy followed by curative surgery. We compared gene expressions that had previously been shown to be involved in chemotherapy or radiation effects; one obtained using RNA extracted from cancer cells by microdissection, and the other from bulky cancer tissues in all patients. More than 50% regression of the primary tumor was seen in 16 patients (40.0%). There was no significant difference in candidate gene expression profiles between tumor and stromal cells except for thymidine phosphorylase (TP). Without microdissection, there was no significant association between distant recurrence and gene expression in specimens. With microdissected sample analysis, however, patients who developed distant recurrence were found to have significantly higher intratumoral thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT) compared with patients without recurrence. It is possible that microdissection is essential for gene expression analysis of clinically irradiated rectal specimens because preoperative chemoradiotherapy for rectal cancer affects the tumor-stroma balance in irradiated rectal cancer specimen.