OBJECTIVES: This study was performed to investigate the detection rate of EGFR T790M mutation by repeated rebiopsy, to identify the clinical factors related to repeated rebiopsy, and to assess survival outcomes according to the methods and numbers of repeated rebiopsies in patients with lung adenocarcinoma who received sequential osimertinib after failure of previous 1st or 2nd generation EGFR-tyrosine kinase inhibitors. METHODS: This retrospective study included patients with advanced-stage lung adenocarcinoma who were confirmed to have EGFR T790M mutation and to have received osimertinib from January 2020 to February 2021 at Samsung Medical Center. The presence of T790M mutation was assessed based on either plasma circulating tumor DNA (ctDNA) or tissue specimens. Results A total of 443 patients underwent rebiopsy, with 186 (42.0%) testing positive for the T790M mutation by the sixth rebiopsy. The final analysis included 143 eligible patients. Progression-free survival was not significantly different in terms of the methods (tissue: 13.3 months, 95% confidence interval [CI]: [9.4, 23.5] vs plasma: 11.1 months, 95% CI: [8.1, 19.4], p = 0.33) and numbers (one: 13.4 months, 95% CI: [9.4, 23.5] vs two or more: 11.0 months, 95% CI: [8.1, 14.8], p = 0.51) of repeated rebiopsies. Longer overall survival (OS) was found in patients in whom T790M was detected by tissue specimens rather than by plasma ctDNA (2-year OS rate: 81.7% for tissue vs 63.9% for plasma, p = 0.0038). Factors related to the lower numbers of rebiopsies included age and bone metastasis. Factor associated with T790M detection in tissue rather than in plasma was pleural metastasis, while advanced tumor stage was related to T790M confirmation in plasma rather than in tissue. CONCLUSIONS: Repeated rebiopsy for T790M detection in patients with NSCLC can increase the detection rate of the mutation. Detection of T790M by plasma ctDNA might be related to poor survival outcomes.