Background: AZD9291, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is active against patients with EGFR T790M-mutant non–small cell lung cancer (NSCLC) who failed prior treatment with EGFR TKIs. However, acquired resistance to AZD9291 is inevitable. In this study, we retrospectively analyzed mechanisms of acquired resistance to AZD9291 in advanced NSCLC. Methods: A total of 293 advanced lung adenocarcinoma patients with signs of AZD9291 resistance were enrolled in the study from January to October 2017. Tissue biopsy was the first choice for mutation profiling, and ctDNA testing was used as an alternative. All samples were analyzed using next-generation sequencing based ER-Seq method, which enables simultaneously assess single-nucleotide variants, insertions/deletions, rearrangements, and somatic copy-number alterations across at least 59 genes (59-1021). Results: At resistance, 3 molecular subtypes emerged: 66 cases (22.5%) lost original EGFR activating mutation and T790M mutation; 130 cases (44.4%) lost the T790M mutation despite detecting of the underlying EGFR activating mutation; 97 cases (33.1%) maintained both EGFR activating mutation and T790M mutation. Known resistance mechanisms detected in 11/66 (16.7%), 56/130 (43.1%), 62/97 (63.9%) cases respectively for all the 3 subtypes. The most frequently known mechanism was EGFR C797S which was identified in 60 cases, while EGFR L792H/V, G796S, L718Q/V, G719A, and E709K were in 27 cases. Activating mutations of PI3K-AKT-mTOR signaling, amplification of the MET, activating mutation / amplification of ERBB2, ALK/ROS1/RET fusion, activating mutation of BRAF, or KRAS were identified in 37, 16, 14, 9, 8, 5 patients respectively. Moreover, 24 cases had Rb1 loss of function mutation. Co-occurrence of resistance mechanisms were observed in 31 patients. Conclusions: There was a high frequency of inter and intra-patient heterogeneity of resistance mechanisms after AZD9291 therapy. Comprehensive NGS analysis may facilitate the broad exploration of potential resistance mechanisms.