Objectives It is clinically important to identify T790M mutation in patients who are suspected to have acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). We therefore aimed to investigate the risk factors of acquired T790M mutation among patients with lung adenocarcinoma with EGFR mutation who were treated using EGFR-TKI, and to identify the clinical impact of re-biopsy. Materials and Methods This is a multicenter, retrospective cohort study in South Korea. From January 2007 to June 2017, patients with adenocarcinoma with EGFR mutation who received re-biopsy and were treated with EGFR-TKI were included in this study. Results A total of 352 patients were included in this study. T790M mutation was identified in 156 (41.9%) study patients at the time of re-biopsy; the median duration from initial biopsy to re-biopsy was 17 months. The univariate logistic analysis showed that exon 19 deletion (odds ratio [OR], 1.643; p = 0.026) and the absence of L858R (OR, 0.627; p = 0.042), and previous EGFR-TKI treatment duration (OR, 1.039; p < 0.001) were associated with T790M mutation. The univariate Cox proportional hazard model showed that brain metastasis at initial diagnosis (hazard ratio [HR], 1.439; p = 0.030) was associated with T790M mutation. Among the patients with T790M mutation at re-biopsy, the osimertinib user group (n = 90) had a better 1-year survival (68.7 vs. 58.3%, p = 0.048) than the osimertinib non-user group (n = 66). Conclusion Re-biopsy might affect the clinical course of patients with EGFR-mutant adenocarcinoma, especially patients with a history of long-term use of EGFR-TKI, exon 19 deletion mutation, and brain metastasis at initial diagnosis