Abstract: Objective To screen differential DNA methylation between esophageal squamous cell carcinoma (ESCC) tisssues and normal esophageal tissues using gene-chip technology, and to construct aberrant methylation panel of tumor suppressor genes in esophageal squamous cell carcinoma. Methods Illumina 450K bead-chip was applied to detect methylation status in ESCC tissues and adjacent tissues from esophageal cancer patients and normal mucosa samples from healthy controls. A total of 485 577 loci sites were analyzed and compared. Aberrant hyper-methylated sites were filtrated according to delta beta value and diffscore, together with functional analysis based on Genecards and Intogen database. Screened genes were validated using mass spectrometry technology. Results A total of 33 713 differential methylated loci were found by comparing ESCC tissues and normal mucosa tissues, including 27 670 hyper-methylated sites, which mainly located in genosome and promoter 5' untranslated region. Considering description and literature reports of biological functions, a panel of aberrant methylation biomarkers was screened out, including TMEFF2，CDH13，ING2，CASZ1，IQGAP2，ADAMTS9，AIM2，TRIT1，KLF6，EBF3, etc. There was no significant difference in the methylation rate of three CG sites in AIM2 gene between cancer tissues and normal controls. CASZ1_CpG_5 together with other four surrounding CG sites showed significant higher methylated frequency in ESCC tissues detected by chip inspection. Conclusion Gene-chip technology could be applied for preliminary screening of differential methylated genes. However, aberrant methylation panel of genes in esophageal cancer should be further validated before being applied as a clinical biomarker.