An adaption of an RNA/RNA duplex, base pair-mismatch assay is capable of detecting rifampin resistance in Mycobacterium tuberculosis. The specificity and sensitivity of the mismatch assay in detecting rifampin resistance were 100% and 96%, respectively, when tested against 46 rifampin-resistant and rifampin-susceptible strains of M. tuberculosis. By use of a range of mycobacterial and nonmycobacterial prokaryote pathogens, the mismatch assay was shown to be specific for M. tuberculosis and Mycobacterium bovis. The assay is cost-effective compared with DNA sequencing and other molecular methods and is simple to perform and interpret. Furthermore, the assay can return a result within 24 h after receipt of an isolated organism and potentially can be used directly with smear-positive specimens.