Background: c-Met amplification and T790M are both recognized as the common molecular mechanisms of acquired resistance (AR) to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in advanced NSCLC. Methods: Advanced NSCLC patients with AR to EGFR TKIs were detected for c-Met overexpression by immunohistochemistry ≥ 50% tumor cells with moderate to high intensity staining were defined as c-Met positive. The statuses of EGFR, ALK, KRAS and ROS1 were tested. c-Met, p-Met, EGFR, p-EGFR, ERBB3, p-ERBB3, AKT, p-AKT, MAPK, p-MAPK, which were important markers in MET and EGFR signal pathway ,were tested by IHC. Results: From January 2013 to January 2015, 126 advanced NSCLC patients with AR to gefitinib or erlotinib were enrolled prospectively. The frequency of c-Met overexpression was 28.6% (36/126), c-Met overexpression+T790M 13.5% (17/126), T790M 24.6% (31/126), SCLC or squamous cell transformation 1.6% (2/126), KRAS mutation 0.8% (1/126), ROS1 fusion 0.8% (1/126) ,ALK fusion 0.8%(1/126) and unknown mechanism 29.3% (37/126), respectively. Eleven c-Met overexpressed patients received gefitinib plus c-Met inhibitors crizotinib. Response rate (RR) by RECEIST was 45.5% (5/11), Disease control rate (DCR) was 54.5% (6/11), Progression disease (PD) was 45.5% (5/11). For patients with c-Met overexpression and without T790M DCR was 100% (5/5). Among these patients 40% (2/5) cases were ongoing at data cut-off. The longest duration of response to date is > 6 months. For patients with both c-Met/T790M positive no RR was found. We detected the protein expression of c-Met, p-Met, EGFR, p-EGFR, ERBB3, p-RBB3, AKT, p-AKT, MAPK, p-MAPK in tumor of 9 cases with c-Met/T790M by IHC. The results showed that all of the markers were positive in ≥ 50% cases (each marker was positive in at least 5 cases, respectively). Conclusions: c-Met overexpression could be as a biomarker for AR. Combination of EGFR TKIs and c-Met inhibitor is a good strategy to overcome AR for c-Met overexpressed patients, but not effective in c-Met/T790M-coexisting cases. One of the possible mechanism of resistance could contribute to both MET and EGFR signal pathway active.