SETTING: Six health facilities in Dar es Salaam, Tanzania.OBJECTIVE: To evaluate the use of stool specimens in the diagnostic workup of paediatric TB using the Xpert^® MTB/RIF assay.DESIGN: Between December 2018 and May 2019, we performed a cross-sectional diagnostic study of children aged between 1 month and 14 years with presumptive TB. A single stool specimen was tested using Xpert. The result was compared with the reference microbiological standard for respiratory or gastric specimens tested using Xpert and/or solid culture. The sensitivity, specificity and predictive values of stool Xpert assay were assessed.RESULTS: A total of 225 children with a median age of 2.17 years (IQR 1.16–5.19) were enrolled; 165/225 (73.3%) were aged <5 years. Of 225 children, 8 (3.6%) were diagnosed with TB as they were culture- or Xpert-positive on sputum/gastric aspirate. The stool Xpert assay showed a sensitivity of 62.5% (95% CI 25–92) and specificity of 100% (95% CI 98–100) against the reference standard.CONCLUSION: Use of the Xpert assay on stool specimens had a moderate sensitivity and high specificity in the diagnosis of pulmonary TB in children. Our data adds to the body of evidence for the use of Xpert assay on stool as a non-respiratory specimen to complement conventional methods used to diagnose the disease.