ABSTRACTTuberculosis remains a major global public concern as a leading cause of health care-associated infections. The detection ofMycobacterium tuberculosis(MTB) is challenging due to the paucibacillary nature of the pathogen. For suspected pulmonary and extrapulmonary tuberculosis patients, if sputum, bronchoalveolar lavage fluid (BALF), related samples are negative for MTB, or suspected tumors, biopsy tissues may provide a better diagnostic yield. This study was aimed at comparing the performances of three methods in identifying MTB in biopsy tissues, including the Bactec mycobacterial growth indicator tube 960 (MGIT 960) system, the GeneXpert MTB/RIF assay (GeneXpert), and the Bactec Myco/F lytic culture (Myco/F) system. Biopsy samples from 3,209 nonduplicated patients were retrospectively enrolled between January 2018 and September 2021, of which 180 (5.6%) were positive for MTB by at least one method. GeneXpert revealed the highest recovery rate (134/162, 82.7%), followed by MGIT 960 (99/135, 73.3%) and Myco/F (26/143, 18.1%), and the composite positive rate for GeneXpert and MGIT 960 was 96.6% (173/179). Pairwise comparisons were conducted after completion of both tests, and the results showed that Myco/F had significantly lower detection rates than GeneXpert and MGIT 960 (16.4% versus 82.8%,P <0.001; 14.3% versus 71.4%,P <0.001). In summary, GeneXpert was the most sensitive and recommended method for MTB detection in biopsy tissues, and the combination of GeneXpert and MGIT 960 could improve the overall diagnostic yield.IMPORTANCEMycobacterium tuberculosis(MTB) poses a severe threat to public health worldwide. The diagnosis of tuberculosis is challenging due to the low load of the microorganism in samples. Biopsy tissues are sometimes collected via invasive procedures with limited size, and additional samples are often inaccessible. The GeneXpert MTB/RIF assay, Bactec MGIT 960 system, and Bactec Myco/F lytic system have been used in detecting MTB in our laboratory. Here, we evaluated the performances of these three methods in 3,209 biopsy tissues samples to establish a more effective protocol based on clinical requirements. Attempts for a locally optimized protocol should be always made.