Background:“Pan-negative” non-small cell lung cancer (NSCLC) patient specimens that lack mutations in known targetable genes. EGFR extracellular domain mutations (ECD) as novel oncogenic mutations are found in colorectal cancer, glioma, and neuroblastoma cases and has not yet reported in NSCLC. No enough evidence between icotinib treatment and ECD has been reported in NSCLC. Methods: Comprehensive mutational analyses were performed on 3279 NSCLC specimens. In this cohort of patients, common lung cancer oncogenic driver mutations were firstly detected in EGFR, as well as the KRAS, BRAF, ALK, ROS1 and RET genes by next generation sequencing tumor DNA (ctDNA) and apply icotinib for treatment for EGFR ECD. Results: Of this entire cohort, sixteen (0.49%) patients (3279 cases) were identified with EGFR ECD, including p.L62R (2), p.R98Q (1), p.I213M (1), p.A237F (1), p.A289V (1), p.A289T (1), p.T302H (1), p.T354K (1), p.T363N (1), p.D368Y (1), p.T430S (1), p.A508V (1), p.N528D (1), p.K593Q (1) and G598V (1). During the treatment by icotinib, two cases (p.A289V, p.A289T) had partial response to icotinib for four and six months, which suggested that icotinib conferred sensitivity to A289X mutation. EGFR A289X mutations were sensitive to icotinib treatment in BaF3 cell lines and in xenograft models. These results paralleled those seen with the well-described EGFR oncogenic driver mutation, L858R, suggesting similar mechanistic underpinnings for the mutations. Conclusions: Here, a new EGFR driver mutation, A289X, was identified in the ECD of two NSCLC specimens. NGS may expand the EGFR mutations spectrum for icotinib treatment in NSCLC, however, it needs to be confirmed in more patients with NSCLC in East Asian and other populations.