To investigate the relationship between the hypermethylation statuses of the mismatch repair genes (hMLH1 and hMSH2) promoters and explore the correlation between it and the development of pancreatic cancer and the biological behavior of pancreatic cancer. We selected 90 patients who were diagnosed with pancreatic cancer and underwent radical operations in the First Affiliated Hospital of the Dalian Medical University between January 2002 and June 2008. The methylation status of the hMLH1 and hMSH2 promoters was detected by methylation-specific polymerase chain reaction (MSP). Meanwhile, the expression of hMLH1 and hMSH2 protein was detected by Western blot and immunohistochemistry, and its correlation with biological behavior of pancreatic cancer was explored. Of the 90 cases, hMLH1 promoter methylation was detected in 54 (60.0%) patients, while none of the paracancerous tissues indicated methylation. In the study, the hMLH1-methylated tumors lost hMLH1 protein expression, but the non-hMLH1-methylated tumors were not seen to have a loss of hMLH1 protein expression (P 0.05). After universal analysis, hMLH1 expression was significantly related to tumor differentiation, lymph node metastasis, and tumor location (P 0.05). Our study suggests that the mismatch repair genes play an important role in pancreatic cancer carcinogenesis and progression through epigenetic modification, and it may be regarded as a potential target for the management of pancreatic cancer.