Lung cancer is the leading cause of cancer deaths worldwide. This study was designed to select multiple DNA markers, which have high sensitivity and specificity to serve as biomarkers for diagnosis of lung cancer. We examined the promoter hypermethylation of three tumor suppressor genes by methylation-specific PCR (MSP), and the instability of eight microsatellite markers by loss of heterozygosity (LOH) and microsatellite instability (MSI) analyses in lung tumor tissues and matched sputum specimens from 79 lung cancer patients. On the basis of the results of sensitivity, specificity, and concordance from each marker analyzed, we selected seven biomarkers, which are LOH of D9S286, D9S942, GATA49D12, and D13S170, MSI of D9S942, and methylation of p16(INK4a) and RARbeta, from the sputum analyses. These selected etiologically associated biomarkers can potentially be used as supplemental diagnostic biomarkers for early lung cancer detection.