To explore the tumor mutation characteristics of patients with non-small cell lung cancer at different lesion locations in the lung and the underlying molecular mechanisms leading to differences in clinical manifestations. High-throughput sequencing was performed on fresh tumor tissue samples from 62 lung cancer patients admitted to our hospital, and the sequencing data of 502 non-small cell lung cancers in the TCGA database were collected, and the characteristics and functional characteristics of gene mutation were analyzed from the perspectives of different lung lobes and distribution sides. Mutations were detected in 99.1% (559/564) of the samples. The proportion of samples with two mutations detected in different groups is the highest(63.01%~82.93%). There were significant differences in the types and frequencies of mutated genes and loci among the groups, but there was no correlation between them and clinical characteristics (age, gender). The functional prediction results show that the non-shared genes between different lung lobe groups are mainly related to the regulation of carbon metabolism, drug resistance in the cancer center, while the non-shared genes between the left and right lung cancer groups are mainly related to the regulation of PD-L1 expression, cell proliferation, mismatch repair and apoptosis signaling pathway, etc. There are significant differences in tissue mutation characteristics and biological function changes in lung cancer patients with different primary sites, which is of great significance for the study of the pathogenesis of non-small cell lung cancer and the auxiliary clinical diagnosis of the primary tumor.