The combined methylation status of the p16, helicase-like transcription factor (HLTF) and T-cadherin, H-cadherin (CDH13) genes in 35 resected primary esophageal squamous cell carcinoma (ESCC) was examined using methylation-specific PCR. To examine whether the methylation status could be a marker for the malignancy of ESCC, the methylation status was correlated with the clinicopathological features of the affected patients. Aberrant methylation of the p16, HLTF and CDH13 genes was detected in 28 (80%), one (3%) and five (14%) out of 35 ESCC specimens, respectively. ESCC with methylation-positive genes showed a trend towards larger tumor size and deeper invasion (p = 0.139 and p = 0.0664, respectively). Thus, the methylation of the p16, HLTF and CDH13 genes is a high malignancy factor in ESCC.