BackgroundNon‐small cell lung cancer (NSCLC) is a common malignant tumor. DNA hypermethylation in the promoter region has been served as a potential molecular marker for several tumors. The goal of the current study was to assess the diagnostic ability of mutL homolog 1 (MLH1) promoter methylation in NSCLC.MethodsA total of 111 NSCLC patients’ paired tissue samples were obtained to explore the association between MLH1 promoter methylation and NSCLC by methylation‐specific polymerase chain reaction (MSP) method. Public databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to verify our findings.ResultsOur results showed a significantly higher MLH1 methylation frequency in tumor tissue samples than their paired adjacent tissues (P = .008). ROC curve indicated that MLH1MSP assay was a sensitive but not a specific method in the diagnosis for NSCLC (sensitivity = 0.964, specificity = 0.135, AUC = 0.550). And the association between the methylation level and clinical characteristics has no statistical significance. TCGA cohort evinced a higher methylation probability in tumor group compared with nontumor group (the mean β value: −0.449 [−0.467, −0.437] vs −0.466 [−0.472, −0.437], P = .011), which was consistent with our results. Meanwhile, an inverse correlation between MLH1 methylation and MLH1 expression was detected in TCGA and GEO databases.ConclusionsThe MSP method for MLH1 methylation was a sensitive but not a specific diagnostic method for NSCLC.