Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.