Purpose: Despite initial benefit from tyrosine kinase inhibitors (TKIs), patients with advanced non-small cell lung cancer (NSCLC) harboring ALK (ALK⁺) and ROS1 (ROS1⁺) gene fusions ultimately progress. Here, we report on the potential resistance mechanisms in a series of patients with ALK⁺ and ROS1⁺ NSCLC progressing on different types and/or lines of ROS1/ALK-targeted therapy.Experimental Design: We used a combination of next-generation sequencing (NGS), multiplex mutation assay, direct DNA sequencing, RT-PCR, and FISH to identify fusion variants/partners and copy-number gain (CNG), kinase domain mutations (KDM), and copy-number variations (CNVs) in other cancer-related genes. We performed testing on 12 ROS1⁺ and 43 ALK⁺ patients.Results: One of 12 ROS1⁺ (8%) and 15 of 43 (35%) ALK ⁺ patients harbored KDM. In the ROS1⁺ cohort, we identified KIT and β-catenin mutations and HER2-mediated bypass signaling as non-ROS1-dominant resistance mechanisms. In the ALK⁺ cohort, we identified a novel NRG1 gene fusion, a RET fusion, 2 EGFR, and 3 KRAS mutations, as well as mutations in IDH1, RIT1, NOTCH, and NF1 In addition, we identified CNV in multiple proto-oncogenes genes including PDGFRA, KIT, KDR, GNAS, K/HRAS, RET, NTRK1, MAP2K1, and others.Conclusions: We identified a putative TKI resistance mechanism in six of 12 (50%) ROS1 ⁺ patients and 37 of 43 (86%) ALK⁺ patients. Our data suggest that a focus on KDMs will miss most resistance mechanisms; broader gene testing strategies and functional validation is warranted to devise new therapeutic strategies for drug resistance. Clin Cancer Res; 24(14); 3334-47. ©2018 AACR.