Pathogenesis of lung cancer is a complicated biological process including multiple genetic and epigenetic changes. Since cigarette smoking is confirmed as the most main risk factor of non-small cell lung cancer (NSCLC), the aim of this study was to determine whether tobacco exposure plays a role in gene methylation. Methylation of the RAR-β gene were detected using methylation-specific polymerase chain reaction in DNA from 167 newly diagnosed cases with NSCLC and corresponding 105 controls. A significant statistical association was found in the detection rate of the promoter methylation of RAR-β gene between NSCLC and controls (x2=166.01; p<0.01), and hypermethylation of the RAR-β gene was significantly associated with smoking status (p=0.038, p<0.05). No relationship was found between RAR-β gene methylation and pathologic staging including clinical stage, cell type, gender and drinking (p>0.05), and the methylation of RAR-β gene rate of NSCLC was slightly higher in stages III+IV (80.0%) than in I+II (70.8%). Similar results were obtained for methylation of the RAR-β gene between squamous cell carcinoma (77.9%) and other cell type lung cancer (73.9%). These results showed that the frequency of methylation increased gradually with the development of clinical stage in smoking-associated lung cancer patients, and tobacco smoke may be play a potential role in RAR-β gene methylation in the early pathogenesis and process in lung cancer, particularly squamous cell carcinoma. Aberrant promoter methylation is considered to be a promising marker of previous carcinogen exposure and cancer risk.