The identification of acquired resistance mutations has been essential in non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) active mutations. Rebiopsy plays a pivotal role in selecting the optimal treatment for patients who develop resistance to initial EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This multicenter, observational study was conducted to investigate the details of rebiopsy in Japanese clinical practice. The primary endpoints were the implementation rate of rebiopsy and the concordance rate for T790M mutation detection between histological and cytological specimens using the cobas EGFR Mutation Test, version 2. One hundred ninety-four patients with EGFR-mutant NSCLC were enrolled, and 120 patients developed acquired resistance to EGFR-TKIs. The median age was 68 years (range 20–87), and 52.5% of the patients were women. Rebiopsy was performed in 109 patients, and the implementation rate of rebiopsy was 90.8%. The success rates of rebiopsy in the total, histology, cytology and liquid biopsy populations were 67.9%, 81.3%, 66.7% and 43.8%, respectively. The positive percent agreement and the negative percent agreement in the detection of the T790M mutation between the histological and cytological specimens were both 90.9%. Obtaining histological or cytological tissue samples at rebiopsy may contribute to improving the detection rate of the T790M mutation (trial registration number: UMIN000026019).