RASSF1A is a novel putative tumor suppressor gene located in 3p21.3 region. The most common inactivation mechanism of RASSF1A is promoter hypermethylation, which is observed in multiple solid tumors including lung cancer. In the present study, we identified the methylation status of RASSF1A in lung cancer sera using methylation-specific PCR and analyzed its clinicopathological significance. Hypermethylation of RASSF1A was detected in 27 of 80 (33.8%) cancer patients but no benign pulmonary disease patients or healthy donors (P<0.001). RASSF1A hypermethylation was preferentially observed in small cell lung cancer (P=0.042), while no statistical difference was found among methylation frequencies of different subtypes of non-small cell lung cancer. RASSF1A methylation status was associated with differentiation (P=0.009) and stage (P=0.013), but not with gender, age or treatment. These findings suggest that serum RASSF1A hypermethylation is a promising molecular biomarker for lung cancer diagnosis.