MicroRNA (miR)-34 and miR-124 have been demonstrated as a potential tumor suppressor in several types of cancers. Each family includes three processed miRNAs (miR-34a, miR-34b, miR-34c, miR-124-1, miR-124-2, and miR-124-3). The tran-scriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. To understand the role of miR-34 and miR-124 in non-small cell lung cancer (NSCLC) and to develop a promising candidate biomarker for diagnosis and prognosis of NSCLC, we determined the methylation status of 6 miR genes in primary lung tissues using a methylation-specific PCR (MSP) and evaluated their relationship with clinicopatholgial features and survival in patients with NSCLC. The methylation frequencies of 6 miR genes were 15.1% for miR-34a, 55.9% for miR-34b/c, 25.6% for miR-124-1, 52.9% for miR-124-3 in malignant tissues. The methylation frequencies of miR-34b/c and miR-124-3 were significantly higher in squamous cell carcinomas than in adenocarcinomas. In addition, miR-34b/c methylation was frequently observed in EGFR mutation-negative adenocarcinomas compared to EGFR mutation-positive ones. However, there was no significant difference in overall survival of the total patients according to six miR genes.