To identify molecular factors that determine duration of response to EGFR tyrosine kinase inhibitors and to identify novel mechanisms of drug resistance, we molecularly profiled -mutant tumors prior to treatment and after progression on EGFR TKI using targeted next-generation sequencing. Targeted next-generation sequencing was performed on 374 consecutive patients with metastatic -mutant lung cancer. Clinical data were collected and correlated with somatic mutation data. Erlotinib resistance due to acquired MTOR mutation was functionally evaluated by and studies. In 200 -mutant pretreatment samples, the most frequent concurrent alterations were mutations in , and and focal amplifications in , and Shorter time to progression on EGFR TKI was associated with amplification of (HR = 2.4, = 0.015) or (HR = 3.7, = 0.019), or mutation in (HR = 1.7, = 0.006). In the 136 posttreatment samples, we identified known mechanisms of acquired resistance: EGFR T790M (51%), (7%), and amplifications (5%). In the 38 paired samples, novel acquired alterations representing putative resistance mechanisms included fusion, fusion, amplification, loss, and an MTOR E2419K mutation. Functional studies confirmed the contribution of the latter to reduced sensitivity to EGFR TKI and -mutant lung cancers harbor a spectrum of concurrent alterations that have prognostic and predictive significance. By utilizing paired samples, we identified several novel acquired alterations that may be relevant in mediating resistance, including an activating mutation in MTOR further validated functionally. .