f Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n ؍ 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosis isolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.T uberculosis (TB) remains a major health concern in both developed and developing countries due to the high rates of morbidity and mortality associated with this disease. In 2010, there were an estimated 8.5 to 9.2 million incident cases of TB globally, with an associated 1.5 million deaths, 23% of which were associated with HIV coinfection (28).The Mycobacterium tuberculosis complex (MTC) comprises eight members (M. tuberculosis, M. canettii, M. africanum, M. bovis, M. caprae, M. microti, M. pinnipedii, and the attenuated M. bovis BCG vaccine strain), ϳ99% similar on a nucleotide sequence level, that are responsible for causing TB in humans (5).While M. tuberculosis is responsible for the majority of cases of human TB, the accurate identification of other MTC members that cause infection is not routinely performed (21). As a result, the global frequency and distribution of each MTC member remain largely unknown, with some studies suggesting that TB caused by members of the MTC other than M. tuberculosis are more prevalent than previously reported (1, 26). M. africanum, for example, has been identified as the causative agent of up to 50% of human TB infections in West African countries (15), and M. canettii was responsible for ϳ10% of TB cases in a recent study performed in the Republic of Djibouti (17). Furthermore, M. bovis remains an important cause of zoonotic TB worldwi...