Lung cancer is the leading causes of cancer-related death worldwide. Precise treatment based on next-generation sequencing technology has shown advantages in the diagnosis and treatment of lung cancer. This cohort study included 371 lung cancer patients. The lung cancer subtype was related to the smoking status and sex of the patients. The most common mutated genes were TP53 (62%), EGFR (55%), and KRAS (11%). The mutation frequencies of EGFR, TP53, PIK3CA, NFE2L2, KMT2D, FGFR1, CCND1, and CDKN2A were significantly different between lung adenocarcinoma and lung squamous cell carcinoma. We identified the age-associated mutations in ALK, ERBB2, KMT2D, RBM10, NRAS, NF1, PIK3CA, MET, PBRM1, LRP2, and CDKN2B; smoking-associated mutations in CDKN2A, FAT1, FGFR1, NFE2L2, CCNE1, CCND1, SMARCA4, KEAP1, KMT2C, and STK11; tumor stage-associated mutations in ARFRP1, AURKA, and CBFB; and sex-associated mutations in EGFR. Tumor mutational burden (TMB) is associated with tumor subtype, age, sex, and smoking status. TMB-associated mutations included CDKN2A, LRP1B, LRP2, TP53, and EGFR. EGFR amplification was commonly detected in patients with acquired lcotinib/gefitinib resistance. DNMT3A and NOTCH4 mutations may be associated with the benefit of icotinib/gefitinib treatment.