BackgroundTo distinguish early‐stage lung cancer from benign disease in pulmonary nodules, especially lesions with ground‐glass opacity (GGO), we assessed gene mutations of ctDNA in peripheral blood using targeted next‐generation sequencing (NGS).MethodsSingle pulmonary nodule patients without mediastinal lymph nodes and symptoms that were hard to diagnose by chest CT and lung cancer biomarker measurement in multiple medical centers were enrolled into the study. All patients accepted minimally invasive surgery but refused preoperative biopsy. Gene mutations in preoperative blood samples were detected by targeted NGS. Mutations with significant differences between lung tumors and benign lesions, as grouped by postoperative pathology, were screened. Protein expression was determined by immunohistochemistry. Highly expressed genes were selected as biomarkers to verify the mutations in peripheral blood.ResultsIn the training set, the RNF213, KMT2D, CSMD3 and LRP1B genes were mutated more frequently in early‐stage lung cancer (27 cases) than in benign nodules (15 cases) (P < 0.05). High expression of the RNF213 gene in lung cancers and low expression in benign diseases were seen by immunohistochemistry. The RNF213 gene was mutated in 25% of lung cancer samples in the validation set of 28 samples and showed high specificity (100%). In GGO patients, RNF213 was mutated more frequently in early‐stage lung cancer compared to benign diseases (P < 0.05).Conclusions RNF213 gene mutations were observed more frequently in early‐stage lung cancer, but not in benign nodules. Mutation of the RNF213 gene in peripheral blood may be a high specificity biomarker for the assisted early diagnosis of lung cancer in pulmonary nodules.Key pointsSignificant findings of the study: In peripheral venous blood and tumor tissue, RNF213 gene mutated more frequently in lung cancer than benign pulmonary nodules.What this study adds: Detection mutation of the RNF213 gene in peripheral blood may be a high specificity method for the assisted early diagnosis of lung cancer in pulmonary nodules.