Background: There are many causes of mediastinal and hilar lymphadenopathy, such as neoplasms, granulomatous diseases, infections and reactive hyperplasia. Nowadays, the popularity of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasing in the diagnosis of mediastinal and hilar lymphadenopathy. We aimed to investigate the diagnostic value of EBUS-TBNA in patients with mediastinal and/or hilar lymphadenopathy and previously conventional TBNA-negative or inadequate results. Methods: Retrospective analysis was performed in 64 patients with previously conventional TBNA-negative or inadequate results and consequently undergoing EBUS-TBNA between July 2007 and August 2011. Results: One hundred and twenty three lymph nodes were sampled by EBUS-TBNA in 64 patients with no complications. In the 63 (98.4%) cases with adequate results, the sensitivity, diagnostic accuracy, and NPV of EBUS-TBNA per patient was 90.5%, 90.6%, and 66.6%, respectively. In a total of 122 (99.1%) adequately sampled lymph nodes, the diagnostic sensitivity, accuracy, and NPV of EBUS-TBNA per nodal station were 87.8%, 90.1%, and 65.7%, respectively. Non-small cell lung cancer (NSCLC) (n = 21, 33.3%) and sarcoidosis (n = 16, 25.3%) were the most common malignant and benign diseases in the patients with adequate samples by EBUS-TBNA. The relationships of diagnostic accuracy with the number of lymph nodes sampled, number of passes per node, or size of lymph nodes were both insignifi cant ( p >0.05). Conclusion: EBUS-TBNA is a sensitive and accurate method for the assessment of mediastinal and hilar lymph nodes in patients with conventional TBNA negative results.