BACKGROUND Epidermal growth factor receptor (EGFR) T790M mutation drives acquired drug resistance to EGFR tyrosine kinase inhibitors (EGFR‐TKIs) in patients with EGFR‐mutant lung cancer. However, it was reported that this mutation may exist before drug exposure. The objective of the current study was to evaluate whether the clinical outcomes are affected by the percentage of preexisting T790M mutations within a tumor. METHODS Pretreatment tissues were collected from 124 patients with advanced non‐small cell lung cancer with sensitizing EGFR mutations that were detected by direct sequencing. Genotyping for EGFR T790M mutation was further performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Patients who were positive for the T790M mutation were divided to 2 subgroups according to T790M mutant signal frequency. RESULTS The T790M mutation was found in 31 patients (25.0%). The T790M mutation frequency at which the risk of disease progression after therapy with EGFR‐TKIs begins to increase was estimated to be 3.2%. The patients with T790M‐positive tumors had a shorter time to disease progression after treatment with EGFR‐TKIs (median, 6.3 months vs 11.5 months; P < .001) and overall survival (median, 16.1 months vs 26.5 months; P = .065) compared with those with T790M‐negative tumors. Among the T790M‐positive patients, the patients with high T790M frequency (9 patients) were found to have a shorter time to disease progression (median, 2.4 months vs 6.7 months; P = .009) and overall survival (median, 9.1 months vs 18.7 months; P = .018) compared with those with low T790M frequency (22 patients). CONCLUSIONS A preexisting EGFR T790M mutation was noted in 25% of patients with EGFR‐mutant lung cancer. Patients with a high T790M mutation frequency had worse clinical outcomes to EGFR‐TKIs than patients with a low T790M mutation frequency. Cancer 2014;120:2090–2098. © 2014 American Cancer Society. The presence of a preexisting T790M mutation is not a rare event in patients with epidermal growth factor receptor‐mutant lung cancer and the frequency of the T790M mutation within a tumor has a critical impact on the tumor's biological characteristics and the clinical outcomes of therapy with epidermal growth factor receptor tyrosine kinase inhibitors.