This report describes the evaluation of the novel MTB-DR-RIF 9G test for the accurate detection and discrimination of Mycobacterium tuberculosis (MTB) and rifampicin-resistant M. tuberculosis (MTB-DR-RIF) in the clinical samples. The procedure included the amplification of a nucleotide fragment of the rpoB gene of the MTB and MTB-DR-RIF strains and their hybridization with the immobilized probes. The MTB-DR-RIF 9G test was evaluated for its ability to detect and discriminate MTB and MTB-DR-RIF strains in 113 known clinical samples. The accuracy of the MTB-DR-RIF 9G test was determined by comparing its results with sequencing analysis and drug susceptibility testing. The sensitivity and specificity of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 95.4% (89.5–98.5) and 100% (69.2–100), respectively. The positive predictive value and negative predictive value of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 100% (85.0–95.9) and 66.7% (38.4–88.18), respectively. Sequencing analysis of all samples indicated that the mutations present in the regions identified with the MTB-DR-RIF 9G assay can be detected accurately.