Patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer derive significant clinical benefit from treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Secondary EGFR mutations such as EGFR T790M commonly lead to resistance to these agents, limiting their long-term efficacy. Irreversible EGFR inhibitors such as CL-387,785 can overcome resistance and are in clinical development, yet acquired resistance against these agents is anticipated. We carried out a cell-based, in vitro random mutagenesis screen to identify EGFR mutations that confer resistance to CL-387,785 using T790M-mutant H1975 lung adenocarcinoma cells. Mutations at several residues occurred repeatedly leading to functional resistance to CL-387,785. These variants showed uninhibited cell growth, reduced apoptosis, and persistent EGFR activation in the presence of CL-387,785 as compared with parental H1975 cells, thus confirming their role in resistance. A screen of alternative agents showed that both an alternative EGFR inhibitor and a cyclin-dependent kinase 4 inhibitor led to significant inhibition of cell growth of the resistant mutants, suggestive of potential alternative treatment strategies. These results identify novel mutations mediating resistance to irreversible EGFR inhibitors and reveal alternative strategies to overcome or prevent the development of resistance in EGFR-mutant non-small cell lung cancers.