Abstract: Aim: Few studies have investigated the clonal architecture of non-small cell lung cancer (NSCLC) and its evolution over time. With increasing evidence for the potential impact of intratumour heterogeneity (ITH) on clinical outcome, and its recognition as a driver of cancer progression, there is an unmet need to elicit the implications of ITH for biomarker validation, therapeutic response, and therefore survival. Methods: In a cohort of 11 primary NSCLC tumours, multi-region whole-exome sequencing at a mean depth of 214x was performed on a total of 37 tumour regions. Single nucleotide variants, indels and copy number data were identified using VarScan2 (v2.3.6). Non-silent mutations were validated using ultra-deep amplicon sequencing (mean depth 1189x). The subclonal composition of each tumor was used to construct phylogenetic trees. Candidate driver genes were selected by referencing large-scale lung cancer or pan-cancer sequencing studies, and the Catalogue of Somatic Mutations in Cancer (COSMIC) gene census. Results: ITH was evident in all tumours, with a median of 33% heterogeneous mutations (range 5–61%). Phylogenetic tree analysis revealed branched tumour evolution, with mutations in potential driver genes occurring both early (trunk) and late (branch). Thirty-five percent (range 8-67%) of mutations in potential driver genes were heterogeneous, and included TP53, EGFR, KRAS, BRAF, ALK and ROS1. Overall, heterogeneous non-silent somatic mutations and copy number aberrations occurred frequently. Conclusions: This study demonstrates spatial and temporal heterogeneity of both driver mutations and copy number events, suggesting that NSCLCs may follow multiple distinct evolutionary pathways simultaneously. Knowledge of these pathways may help identify novel therapeutic targets to improve clinical outcomes, and large-scale longitudinal genomic studies, such as TRACERx (TRAcking NSCLC Evolution through therapy (Rx), NCT01888601), may become a central component to the delivery of precision cancer medicine.