Background: Re-biopsy is paramount for further treatment of lung cancer patients with recurrent or progressive disease. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an important method for tissue sampling of mediastinal lesions. However, the role of EBUS-TBNA in re-biopsy of lung cancer is not clear. In this study, we investigated the feasibility of re-biopsy by EBUS-TBNA of pretreated lung cancer patients. Methods: Consecutive patients with pretreated lung cancer with suspected progression or recurrence that underwent EBUS-TBNA between October 2015 and December 2019 were enrolled. The diagnostic yield, specimen adequacy, and complications were assessed. Results: A total of 72 lesions from 45 patients with suspected lung cancer recurrence (n=11) or progression (n=34) were sampled by EBUS-TBNA. The diagnostic yield and specimen adequacy rate was 73.3% (33/45) and 95.6% (43/45), respectively. There was no major complication, but 7 (15.6%) minor complications were noted in these patients. Twenty-one patients had EGFR testing results from both the initial diagnosis and from re-biopsy specimens. The acquired T790M mutation was identified in 5 (38.5%) of 13 patients with EGFR-mutant lung adenocarcinoma. Conclusion: EBUS-TBNA is a feasible and safe method of re-biopsy for pretreated lung cancer patients.