To investigate the impact of oncogenic genetic alterations (GAs) on non-small-cell lung cancer (NSCLC) in southwestern China.We first collected 579 pathologically confirmed NSCLC specimens and then used next-generation sequencing (NGS) to evaluate the DNA samples for GAs. Both the tissue and plasma samples were provided by 28 patients. Furthermore, subgroup analyses based on sample type, concordance, and GA type were carried out.GAs were detected by NGS in 61.8% (358/579) of patients. Two hundred and twenty-nine patients (39.6%) harbored EGFR mutations, 63 (10.9%) harbored KRAS mutations, 13 (2.2%) harbored BRAF mutations, 30 (5.18%) harbored ALK fusions, and 13 (2.2%) had ROS1 fusions. We found that females (p < 0.01), nonsmokers (p < 0.001), adenocarcinoma (p < 0.001), and tissue (p = 0.03) had a relatively high EGFR mutation rate. Notably, NSCLC patients from Xuanwei had a significantly different mutational pattern for EGFR in comparison with that of non-Xuanwei patients (higher G719X + S768I mutations and multiple gene alterations, but fewer exon 19 deletion mutations and single gene alterations). We found that adenocarcinoma (p = 0.02), family history of malignancy (p = 0.03), Xuanwei origin (p < 0.001), and tissue (p = 0.04) were associated with a higher number of KRAS mutations. Subgroup analysis showed that ALK (p < 0.001) and ROS1 (p < 0.05) fusions and rare EGFR mutations (p < 0.001) were associated with non-Han ethnic patients.Yunnan NSCLC patients from Xuanwei and non-Han ethnic patients had an obviously unique prevalence of GAs.© 2020 Ma et al.