Objective: To detect the promoter methylation status of hMLH1 gene in colorectal cancer (CRC) and evaluate its correlation with clinicopathological features and prognosis. Methods: The methylation status of hMLH1 gene was determined using manual microdissection followed by methylation-specific PCR(MSP) in 68 paired CRC specimens and adjacent normal tissues,all of the patients received 4~6 cycles of 5-fluorouracil (5-Fu) based adjuvant chemotherapy. Results: Methylation frequency of hMLH1 in cancerous tissue was 23.5% (16/68),which was significantly higher than that in the corresponding normal tissue (3/68,4.4%, P = 0.001). Although hMLH1 methylation was not associated with patients’ clinicopathological features,Kaplan-Meier analysis showed the methylation state of hMLH1 predicted beneficial progression-free survival (PFS) and overall survival (OS) in CRC (P = 0.039 and P = 0.045,respectively). Conclusion:hMLH1 gene methylation maybe a useful biomarker related to favourable prognosis in CRC patients who received 5-Fu based adjuvant chemotherapy.