Runx3 and CHFR genes were defined as tumor suppressor genes in gastric cancer (GC) recently. This paper was to investigate the roles of methylation and expression status of Runx3 and CHFR genes in GC patients. Methylation-specific polymerase chain reaction (MSP) and bisulfite DNA sequencing (BSP) were used to detect methylation status of Runx3 and CHFR genes in GC patients. The expression of Runx3 and CHFR in GC patients was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analysis. The expression of the protein and mRNA decreased remarkably in the patients with aberrant promoter methylation of Runx3 and CHFR genes. The methylation status of Runx3 and CHFR were inversely related to the tumor size, tumor invasion depth and tumor differentiation in GC patients. Moreover, the protein expression of Runx3 and CHFR were significantly correlated with tumor invasion depth and tumor differentiation, respectively. Aberrant promoter methylation of Runx3 and CHFR genes may be involved in the carcinogenesis and development of GC and may provide useful clues for the prediction of the malignant behaviors of GC.