This study aimed to evaluate the diagnostic value of rapid simultaneous RNA amplification and testing for tuberculosis (SAT-TB) in smear-negative pulmonary tuberculosis (PTB). We performed a multicenter prospective analysis of 206 patients with smear-negative suspected PTB between December 2018 and March 2022. We collected sputum or bronchoalveolar lavage fluid (BALF) for simultaneous SAT-TB and Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assays. The efficiency of SAT-TB detection was also evaluated. The final analysis included 161 patients with smear-negative suspected PTB, of whom 114 provided sputum specimens and 47 provided BALF specimens. In sputum samples, the area under the curve, sensitivity, and specificity of SAT-TB for diagnosing PTB were 0.75, 50.7%, and 100.0%, respectively, and those of the Xpert MTB/RIF assay were 0.81, 62.3%, and 100.0%, respectively. The kappa coefficient k of the consistency between SAT-TB and Xpert MTB/RIF in sputum specimens was 0.686. In BALF specimens, the area under the curve, sensitivity, and specificity of SAT-TB for diagnosing PTB were 0.79, 57.1%, and 100.0%, respectively, and those of Xpert MTB/RIF were 0.86, 76.2%, and 96.2%, respectively. The kappa coefficient k of the consistency between SAT-TB and Xpert MTB/RIF in BALF specimens was 0.656. The SAT-TB and Xpert MTB/RIF assays were highly consistent in diagnosing smear-negative PTB. It is a valuable method for early detection, prevention, and managing smear-negative PTB suspects. Meanwhile, the detection efficiency and cost-effectiveness of SAT-TB are more suitable for the rapid diagnosis of smear-negative PTB in low- and middle-income countries.Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.