Lung cancer is the leading cause of cancer deaths worldwide and is usually associated with late diagnosis and poor prognosis. Tumor-acquired methylation of the promoter CpG islands (CGIs) is an important mechanism for silencing tumor suppressor genes. The checkpoint kinase 2 (CHK2) is a tumor suppressor that plays a crucial role in regulating cell-cycle checkpoints and apoptosis following DNA damage. The methylation statuses of two CGIs, distal and proximal, of human CHK2 gene were determined in non-small cell lung cancers (NSCLCs) using a nested methylation-specific PCR and bisulfite sequencing. The methylation of distal CHK2 CGI was found in 39 (28.1%) of the 139 NSCLCs. Its frequency was significantly more frequent in squamous cell carcinomas than in adenocarcinomas (40.0% vs 19.0%, p=0.006) and was also higher in ever-smokers than in never-smokers with a borderline significance (31.7% vs 17.1%, p=0.071). RT-PCR analysis showed that the distal CGI methylation correlated with CHK2 mRNA expression. However, the methylation of the proximal CHK2 CGI is not specific to tumors and not related to gene expression. These results suggest that the down-regulation of CHK2 gene via distal CGI methylation may play a role in the pathogenesis of NSCLC, particularly squamous cell carcinoma. However, further studies with large numbers of patients are needed to confirm our findings.