To determine the relevance of O-6-methylguanine-DNA methyltransferase (MGMT), human mutS homolog 2 (hMSH2), and human mutL homolog 1 (hMLH1) in TP53 mutations in esophageal squamous cell carcinoma, we employed methylation-sensitive high-resolution melting technology and methylation-specific polymerase chain reaction (PCR) to analyze promoter hypermethylation of MGMT, hMSH2, and hMLH1, respectively, in 51 paired tumors and their adjacent normal tissues. The protein expression of the three proteins was also evaluated by Western blot analysis, and the PCR products of TP53, from exon 5 to exon 8, were directly sequenced to measure the mutation spectrum. Esophageal tumor tissues embraced statistically higher MGMT and hMSH2 promoter methylation level than normal tissue. The promoter methylation status of MGMT and hMSH2 corresponds positively with the protein expression level of MGMT and hMSH2. However, such relevance was not found for hMLH1. Furthermore, TP53 mutation status was well associated with MGMT and hMSH2 promoter methylation status, indicating that silencing of the two genes could lead to TP53 mutation in ESCC.