Background and Aims: The aims are to investigate whether repeat re-biopsy raise the positive rate of T790M and to compare the efficacy of osimertinib between in the first rebiopsy and repeat re-biopsy in patients with epidermal growth factor receptor (EGFR) mutation positive advanced non-small cell lung cancer (NSCLC).Methods: We retrospectively assessed the efficacy repeat rebiopsy and osimertinib in 49 patients to undergo re-biopsy after disease progression of the first or the second generation EGFR tyrosine kinase inhibitor (TKI).Results: Of 49 patients, 26 were male, and the median age was 71 (range, 50-89 years).The type of EGFR mutation was as follows: 23 patients had exon 19 deletion, 23 had exon 21 L858R, and 3 had others.The positive rate of T790M was 43% (17/40) in tissue and 32% (12/38) in plasma.Although the positive rate of T790M in the first re-biopsy was 31%, repeat re-biopsy raised 59%.The response rate, median time to progression, median survival time of osimertinib were 60.0%, 18 months, and 45 months in patients with the first re-biopsy positive, and 69.2%, 10 months, and 59 months in patients with the second or after re-biopsy positive, respectively.There were no differences in response rates, progression free survivals, and overall survivals between patients in the first re-biopsy positive and the second or after re-biopsy positive. Conclusion:The repeat re-biopsy raised the positive rate of T790M from 31% to 59%.