In this prospective multicenter study, we aimed to evaluate the diagnostic accuracy of the novel GeneReady MTB assay on sputum for the detection of pulmonary tuberculosis (TB). Participants with symptoms suggestive of active pulmonary TB from three Chinese hospitals underwent testing with GeneReady, smear microscopy, mycobacterial culture, and Xpert MTB/RIF (G4) assay. GeneReady demonstrated an analytic limit of detection (LOD) of 23.34 CFU/mL (95% confidence interval [95% CI]: 16.96-43.05). A total of 537 participants meeting the inclusion criteria were included in our analysis. Against a microbiological reference standard, GeneReady had a sensitivity of 92.46% (319/345, 95% CI: 89.02%-94.92%) and a specificity of 96.35% (185/192, 95% CI: 92.33%-98.39%). When stratified by bacterial load, the sensitivity of GeneReady was higher in smear-positive (96.86%) versus smear-negative (87.01%) patients. When compared with Xpert MTB/RIF (G4), GeneReady showed a positive percent agreement of 94.55% (95% CI: 91.48%-96.73%) and a negative percent agreement of 93.24% (95% CI: 88.68%-96.11%). To conclude, GeneReady showed diagnostic accuracy comparable to Xpert MTB/RIF (G4). Its rapid turnaround, lower cost, and ease of use support its potential as an initial diagnostic tool for pulmonary TB in resource-limited settings. Further studies are warranted to evaluate its diagnostic accuracy in clinical settings across different patient populations.IMPORTANCETuberculosis (TB) remains a global health crisis, hindered by diagnostic gaps in resource-limited settings where high costs restrict access to accurate molecular testing. This study demonstrates that the novel GeneReady MTB assay provides diagnostic accuracy comparable to the widely used Xpert MTB/RIF (G4) assay, but at a significantly lower cost and with simplified operation. By offering a rapid, affordable, and accurate alternative, this test has the potential to expand access to quality TB diagnosis in primary and community healthcare settings, thereby improving case detection and supporting global TB control efforts.