Objective: To investigate the expression of epidermal growth factor receptor (EGFR) gene, Kirsten mouse sarcoma (KRAS) gene, anaplastic lymphoma kinase (ALK) gene and mouse sarcoma virus oncogene homolog B1 (BRAF) gene and their clinicopathological characteristics in non-small cell lung cancer (NSCLC) patients. Methods: The clinicopathological data of 396 patients with pathologically confirmed NSCLC in the First Affiliated Hospital of Zhengzhou University were collected. The mutation status of EGFR, KRAS, ALK and BRAF genes in tumor tissues were detected by high-throughput next-generation sequencing (NGS), and the gene mutation rate and its relationship with clinicopathological characteristics were analyzed. Results: The positive rate of EGFR gene mutation was 49.24% (195/396), and the mutation rate was higher in female, adenocarcinoma and non-smoking patients (P<0.05). The positive rate of KRAS gene mutation was 8.59% (34/396), and the mutation rate was higher in male, adenocarcinoma and smoking patients over 60 years old (P<0.05). The positive rate of ALK gene mutation was 6.06% (24/396), and the mutation rate was higher in young patients under 60 years old. The positive mutation rate of BRAF gene was 3.28% (13/396). Among them, there were 1 case of EGFR combined with ALK mutation coexistence, 2 cases of EGFR combined with BRAF mutation coexistence, 1 case of KRAS combined with ALK mutation coexistence, 1 case of ALK combined with BRAF mutation coexistence, 1 case of KRAS combined with BRAF mutation coexistence, and 1 case of EGFR and KRAS combined with BRAF mutation coexistence. Conclusion: The positive rate of EGFR, KRAS, ALK and BRAF gene mutations in NSCLC patients is similar to that reported at home and abroad, and is related to the clinical and pathological characteristics of patients. In some cases, two or more gene mutations can coexist, and the clinical treatment of patients with coexisting mutations still needs to be further studied to provide evidence-based evidence.