The purpose of this study was to detect the methylation of the RUNX3 gene promoter in non-small cell lung cancer (NSCLC) tissue and to explore the association of this methylation with clinical features of NSCLC. In 58 samples of NSCLC tissue and normal adjacent tissue, methylation of the RUNX3 gene promoter was measured by methylation-specific polymerase chain reaction. Correlation with clinicopathological characteristics was assessed. The results demonstrated that RUNX3 gene promoter methylation was present in 26/58 (44.8%) of NSCLC tissue samples and 10/58 (17.2%) of normal tissue samples, and that the difference was statistically significant between the two groups (χ(2)=10.311, p=0.001). Significantly, methylation of the RUNX3 gene promoter correlated with clinical stage, lymph node metastasis and the degree of differentiation (p<0.05) but not with age, gender, smoking history and pathological type (p>0.05). In conclusion, methylation of the RUNX3 gene promoter had a high relevance ratio in NSCLC tissue and correlated with clinical stage, lymph node metastasis and degree of differentiation; thus, this association may have clinical significance in NSCLC.