Pulmonary tuberculosis diagnosis is difficult when patients cannot produce sputum. Most sputum is swallowed, and tuberculosis DNA can survive intestinal transit. We therefore evaluated molecular testing of stool specimens for detecting tuberculosis originating from the lungs. Paired stool and sputum samples (n=159) were collected from 89 patients with pulmonary tuberculosis. Control stool samples (n=47) were collected from patients without tuberculosis symptoms. Two techniques for DNA extraction from stool samples were compared, and the diagnostic accuracy of the PCR in stool was compared with the accuracy of sputum testing by PCR, microscopy, and culture. A heminested IS6110-PCR was used for tuberculosis detection, and IS6110-PCR-positive stool samples then underwent rifampin sensitivity testing by universal heteroduplex generator PCR (heteroduplex-PCR) assay. For newly diagnosed pulmonary tuberculosis patients, stool IS6110-PCR had 86% sensitivity and 100% specificity compared with results obtained by sputum culture, and stool PCR had similar sensitivities for HIV-positive and HIV-negative patients (P=0.3). DNA extraction with commercially available spin columns yielded greater stool PCR sensitivity than DNA extraction with the in-house Chelex technique (P=0.007). Stool heteroduplex-PCR had 98% agreement with the sputum culture determinations of rifampin resistance and multidrug resistance. Tuberculosis detection and drug susceptibility testing by stool PCR took 1 to 2 days compared with an average of 9 weeks to obain those results by traditional culture-based testing. Stool PCR was more sensitive than sputum microscopy and remained positive for most patients for more than 1 week of treatment. In conclusion, stool PCR is a sensitive, specific, and rapid technique for the diagnosis and drug susceptibility testing of pulmonary tuberculosis and should be considered when sputum samples are unavailable.