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Background: The aim of this study was to evaluate the role of Xpert MTB/RIF assay in the detection of Mycobacterium tuberculosis for differentiating tuberculosis intrathoracic lymphadenopathy from sarcoidosis intrathoracic lymphadenopathy. Methods: The patients who were suspected to having sarcoidosis or tuberculosis intrathoracic lymphadenopathy at the Shanghai Pulmonary Hospital between October 1, 2020 and June 30, 2021 were retrospectively evaluated in this study. All patients underwent endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and Xpert analysis. Differences in clinical and radiological features were recorded. The diagnostic performances of EBUS-TBNA Xpert, acid-fast bacilli, culture, and peripheral blood QuantiFERON-TB Gold (QFT) for differentiating sarcoidosis from tuberculosis intrathoracic lymphadenopathy were analyzed. Results: A total of 119 patients were included in this analysis. Of those, 83 patients were finally diagnosed with sarcoidosis (N = 50) and tuberculosis (N = 33) intrathoracic lymphadenopathy. Young individuals were more likely to have tuberculosis versus sarcoidosis intrathoracic lymphadenopathy (P = 0.006). Markers of inflammation, including fever, leukocytes, and serum ferritin levels, were significantly higher in tuberculosis versus sarcoidosis intrathoracic lymphadenopathy (P < 0.01). Bilateral lung involvement and symmetry intrathoracic lymphadenopathy were more common in sarcoidosis intrathoracic lymphadenopathy (P < 0.01). In addition, the longest diameter of intrathoracic lymphadenopathy (in cm) was significantly larger in sarcoidosis intrathoracic lymphadenopathy (P = 0.001). However, the largest diameter of lung lesions was significantly shorter (P = 0.005). The sensitivity and specificity values of Xpert and QFT for differentiating these two diseases were 69.70% and 100%, and 96.43% and 91.84%, respectively. Conclusion: Xpert MTB/RIF is recommended for the diagnosis of tuberculosis intrathoracic lymphadenopathy using EBUS-TBNA samples. A negative QFT suggests the exclusion of the diagnosis of tuberculosis intrathoracic lymphadenopathy. [ABSTRACT FROM AUTHOR]