Objective To study the driver gene detection rate of different sample types in patients with non-small cell lung cancer (NSCLC), the abnormal driver genes of epidermal growth factor receptor (EGFR), anergic lymphoma kinase (ALK), C-ros prooncogene1-receptor tyrosine kinase (ROS1), Kristen mouse sarcoma virus prooncogene homology (KRAS), murine sarcoma viral toxotoxin oncogene homology B (BRAF), and their clinicopathology characteristic relationship. Methods A total of 180 NSCLCs from 909th hospital from January 2021 to December 2021 were collected for driver genes testing including 25 surgical resection specimens, 80 lung biopsy specimens, 29 malignant pleural effusion cell wax block specimens and 46 metastatic lesion specimens. The specimens were tested for driver gene mutations using the amplification refractory mutation system (ARMS) method. Results The total mutation rate of the five driver genes was 53.33% (96/180), and the mutations of the driver genes could be detected in different specimen types, and the difference in mutation rate was not statistically significant (P>0.05). The mutation rate of EGFR gene was 40.56% (73/180), and the difference in gender and tissue type was statistically significant (P<0.05), and the difference in age mutation rate was not statistically significant(P>0.05). KRAS gene mutation rate was 6.11% (11/180), of which the difference between gender was statistically significant (P<0.05), and the difference between age and tissue type mutation rate was not statistically significant (P>0.05). ALK fusion gene mutation rate was 4.44/% (8/180), and the difference between age, gender and tissue type mutation rate was not statistically significant (P> 0.05).ROS1 fusion gene mutation rate was 1.11% (2/180), and BRAF gene mutation was rate 1.11% (2/180). Conclusion Driver genes can be detected in different specimen types, increasing the sampling pathway for clinical genetic testing. The EGFR gene mutation rate was significantly higher in NSCLC patients than in other driver genes, EGFR gene mutations are different from patients′ gender and pattern of organization. KRAS gene mutations are different from patients’ gender. The mutation rate of ALK, ROS1 and BRAF genes is low, but it is of great significance to guide the treatment of NSCLC. Multigene combined testing can obtain more information about driver gene mutations at a time, which can guide clinical treatment faster and more comprehensively