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Objectives: To assess the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), against smear microscopy and culture method for diagnosis of MTB infection. Methods: This is a retrospective analysis of 103 respiratory and 137 non-respiratory patient specimens suspected of tuberculosis at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia performed between April 2014 and March 2015. Each sample underwent smear microscopy, mycobacterial culture, and GeneXpert MTB/RIF test. Results: Fifteen out of 103 respiratory samples were smear and culture positive, whereas 9 out of 137 non-respiratory samples were smear positive. Out of 9 smear positive specimens, 8 were also culture positive. All 15 culture positive respiratory samples were detected by Xpert MTB/RIF (sensitivity and positive predictive value [PPV]=100%). Similarly, all 8 culture positive non-respiratory specimens were identified by Xpert MTB/RIF (sensitivity 100%; PPV 88.8%). The Xpert MTB/RIF detected only one false positive result in 88 smear negative respiratory specimens (specificity 98.9%; negative predictive value [NPV]= 100%). All 125 smear negative non-respiratory specimens tested negative by culture and Xpert MTB/RIF (sensitivity, specificity, PPV, NPV= 100%). Conclusion: The performance of Xpert MTB/RIF was comparable to the gold standard culture method for identification of MTB in both respiratory and non-respiratory clinical specimens. Saudi Med J 2016; Vol. 37 (12): 1404-1407 doi: 10.15537/smj.2016.12.15506