The positive surgical margins are associated with postsurgical recurrence in colorectal carcinoma patients, and molecular margin analysis is considered to be more sensitive in detecting preneoplastic lesions than is conventional histological margin examination. Here, we developed a microarray and established six calibration curves for hMLH1 gene methylation patterns analysis in 20 colorectal resected margin specimens and corresponding tumor tissue specimens as well as four normal tissue specimens. The results indicated that a moderate methylation level (8-42%) was found in 20 surgical margin tissues, extensive methylation (25-58%) was detected in 20 tumor tissues, and little or no methylation was observed in normal tissues. Of the six paired probes, the average methylation levels in 20 tumor tissues were 60, 35, 43, 53, 38, and 27%, whereas the average methylation levels of the six paired probes in 20 surgical margin tissues were 43, 16, 24, 28, 21, and 11%. Thus, this study demonstrated the feasibility of this assay for molecular assessment use. In addition, it will contribute significant information to our understanding of CpG island methylation for cancer diagnosis and postoperative recurrence.