A total of 110 primary NSCLCs (non-small cell lung cancers) were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation-specific PCR (MSPCR) as well as bisulfite genomic sequencing (BGS), and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of (41.2±3.7)%. Significant associations between 3p21 LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyses shows significant correlations from one another. Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significant correlation was also observed in extent between methylation and RASSF1A expression, illustrating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions. A total of 110 primary NSCLCs (non-small cell lung cancers) were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation- specific PCR (MSPCR) as well as bisulfite genomic sequencing (BGS), and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of (41.2 ± 3.7)%. LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyzes shows significant correlations from one another Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significa nt correlation was also observed in extent between methylation and RASSF1A expression, indicating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions.