Acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is a critical issue that needs to be overcome in the treatment of patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. EGFR and AKT are client proteins of the 90-kDa heat shock protein (Hsp90). Therefore, it was hypothesized that the use of Hsp90 inhibitors might allow the resistance to EGFR-TKIs to be overcome. Furthermore, Hsp90 inhibitors are known to function as radiosensitizers in various types of cancer. In the present study, we evaluated the radiosensitizing effect of the novel Hsp90 inhibitor, NVP-AUY922 (AUY), on NSCLC cell lines harboring EGFR activating mutations and showing acquired resistance to EGFR-TKIs via any of several mechanisms. We used HCC827 and PC-9, which are NSCLC cell lines harboring EGFR exon 19 deletions, and gefitinib-resistant sublines derived from the same cell lines with T790M mutation, MET amplification or stem-cell like properties. AUY was more effective against the gefitinib-resistant sublines with T790M mutation and MET amplification than against the parental cell lines, although the subline with stem cell-like properties showed more than a 10-fold higher resistance to AUY than the parental cell line. AUY exerted a significant radiosensitizing effect on the parental cell line and the MET-amplified subline through inducing G2/M arrest and inhibition of non-homologous end joining (NHEJ). In contrast, the radiosensitizing effect of AUY was limited on the subline with stem cell-like properties, in which it did not induce G2/M arrest or inhibition of NHEJ. In conclusion, combined inhibition of Hsp90 plus radiation was effective, and therefore a promising treatment alternative for overcoming major EGFR-TKI resistance, such as that induced by T790M mutation or MET amplification. However, other approaches are required to overcome minor resistance to EGFR-TKIs, such as that observed in cells with stem cell-like properties.