Botswana is experiencing a growing threat of multidrug/rifampicin resistant tuberculosis in recent years. The inability to rapidly diagnose tuberculosis and associated drug resistance may lead to increased morbidity and mortality in patients as well as the spread of resistant strains of tuberculosis to uninfected population. In response to the long turnaround time inherent with conventional drug susceptibility testing methods, Botswana National Tuberculosis Programme adopted the World Health Organization- endorsed Xpert MTB/RIF for the rapid detection of tuberculosis and drug resistance as an alternative to conventional culture-based drug susceptibility testing. This molecular assay allows for early diagnosis of drug resistance and prompt initiation of appropriate therapy. Therefore the aim of this study was to investigate the potential of Xpert MTB/RIF assay to rapidly diagnose drug resistance in routine clinical practice in Botswana and to compare the cost of detecting multidrug/rifampicin resistance with conventional drug susceptibility testing. The findings of this study are critical for disease control and may complement a well-functioning TB control program.