Abstract: Lung cancer is the most common cancer worldwide, and a leading cause of cancer-related death. Despite improvements in molecular diagnosis and targeted therapies, the 5-years overall survival remains poor. To obtain a better insight into the genetic architecture of the most common type of lung cancer, non-small lung cancer (NSCLC), we performed multi-region DNA sequencing on 7 resected NSCLC samples. Ultra-deep sequencing of a comprehensive cancer gene panel and whole-exome sequencing revealed intratumor heterogeneity in all samples, with several putative tumor driver mutations present in some but not all regions of a tumor. Phylogenetic tree analyses based on non-synonymous mutations revealed a branched evolution pattern in all tumors. An adenosquamous tumor showed striking intratumor heterogeneity, with only a third of all non-synonymous mutations shared across all tumor regions, and clear separation of adenocarcinoma and squamous cell carcinoma tumor regions in the remaining two-third of the mutations. Our multi-region exome sequencing data also revealed regional differences in DNA copy number alterations. Some tumors, relatively homogeneous in terms of mutations, displayed high levels of intratumor heterogeneity in terms of DNA copy number changes, indicating the presence of distinct patterns of intratumor heterogeneity that might contribute to disease progression in different tumours. Overall, our multi-region deep exome sequencing data revealed intratumor heterogeneity in NSCLC, demonstrating branched evolution, both in terms of non-synoymous mutations and DNA copy number alterations, which has important implications for our understanding of the clonal evolution of NSCLC.