Non-small cell lung cancer (NSCLC) is featured with complex genomic alterations. Molecular profiling of large cohort of NSCLC patients is thus a prerequisite for precision medicine. We first validated the detection performance of a next-generation sequencing (NGS) cancer hotspot panel, OncoAim, on formalin-fixed paraffin-embedded (FFPE) samples. We then utilized OncoAim to delineate the genomic aberrations in Chinese NSCLC patients. Overall detection performance was powerful for mutations with allele frequency (MAF) ≥ 5% at >500 × coverage depth, with >99% sensitivity, high specificity (positive predictive value > 99%), 94% accuracy and 96% repeatability. Profiling 422 NSCLC FFPE samples revealed that patient characteristics, including gender, age, lymphatic spread, histologic grade and histologic subtype were significantly associated with the mutation incidence of EGFR and TP53. Moreover, RTK signaling pathway activation was enriched in adenocarcinoma, while PI(3)K pathway activation, oxidative stress pathway activation, and TP53 pathway inhibition were more prevalent in squamous cell carcinoma. Additionally, novel co-existence (e.g., variants in BRAF and PTEN) and mutual-exclusiveness (e.g., alterations in EGFR and NFE2L2) were found. Finally, we revealed distinct mutation spectrum in TP53, as well as a previously undervalued PTEN aberration. Our findings could aid in improving diagnosis, prognosis and personalized therapeutic decisions of Chinese NSCLC patients.Lung cancer is the leading cause of cancer-associated mortality worldwide. Up to 90% lung cancer is non-small cell lung cancer (NSCLC) 1 . NSCLC is usually diagnosed at the advanced stage with limited therapeutic options (e.g., surgery, radiotherapy and chemotherapy) 1 .Conventional molecular detection techniques, such as fluorescence in situ hybridization and Sanger sequencing, only detect a limited number of biomarkers 2-4 . The advent of next-generation sequencing (NGS) has broadened the landscape of genetic aberrations 5-7 , making it possible to implement targeted treatment tailored for specific mutations in individual patients 1,8,9 . Lung cancer is characterized with complex genomic aberrations 10 . The most frequently mutated genes in NSCLC include EGFR (Epidermal growth factor receptor), TP53 (Tumor Protein p53), KRAS (Kirsten rat sarcoma viral oncogene) and PIK3CA (Phosphatidylinositol 3-kinase). In addition, Genetic mutations detected in NSCLC are complicated by patient demographic, racial, clinical and pathological characteristics 1,8,9,11 . For instance, the mutational frequency of EGFR can vary from 10% in Western populations to 30% in Asians, and KRAS mutation incidence in Western populations is approximately 18-26% versus 3.8-8% in Asians 12 , and within