Rapid assays are still needed to detect rifabutin (RFB) susceptibility for proper tuberculosis treatment. To assess the use of the GenoType MTBDRplus assay and subsequent rpoB gene sequencing on detection of RFB susceptibility, we analyzed 800 multidrug-resistant Mycobacterium tuberculosis isolates, and 13% (104/800) were RFB susceptible. Of the 104 RFB-susceptible isolates, 71 (68.3%) isolates were rapidly identified using two molecular assays, while the remaining isolates could be determined using conventional drug-susceptibility testing according to the clinician's decision.