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Background: The present study analyzed the relationship between clinical features and the T790M mutation in non-small cell lung cancer (NSCLC) patients resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. Methods: NSCLC patients with resistance to first-generation EGFR-TKIs in which the disease control time was more than 6 months after initial TKI treatment were enrolled. T790M mutation analysis was performed using one of the following methods according to each manufacturer's protocols: Cobas EGFR mutation test (41/105, 39.0%), digital PCR (42/105, 40.0%) or Scorpion amplification refractory mutation system (ARMS) (22/105, 21.0%). Sample type of T790M was from tissue only (53/105, 50.5%), plasma only (46/105, 43.8%), tissue and plasma (6/105, 5.7%). Results: Of 105 patients, 57 were T790M-positive and 48 were T790M-negative. T790M-positive patients had longer progression-free survival (PFS) after initial EGFR-TKI treatment (p = 0.019). T790M positivity was more frequent in patients treated with gefitinib than in those treated with icotinib (65% vs 40.54%, p = 0.018). The rate of T790M positivity was lower in patients with EGFR L858R (44.44%, 12/27) before TKI treatment than in those with EGFR 19del (72.0%, 36/50, p = 0.036). Patients who achieved PR after initial EGFR-TKI treatment had a higher rate of T790M positivity than those with SD (75.76% vs 50%, p = 0.023). There was no relationship between T790M status and age, gender, primary site, metastasis site, or treatment before TKI. Conclusion: Progression-free survival (PFS), drug type, response to initial EGFR-TKI treatment, and EGFR status before initial EGFR treatment were associated with the frequency of T790M mutation. [ABSTRACT FROM AUTHOR]