The objective of this study is to correlate rpoB mutations found on the new-generation sequencing (NGS) in Mycobacterium tuberculosis (MTB) isolates with minimum inhibitory concentrations (MICs) to the rifampicin (RIF). We assessed the minimum inhibitory concentrations for 151 archived clinical MTB isolates that were determined phenotypically susceptible to RIF (101; 66.89%), and the remaining 50 (50; 33.11%) were resistant to RIF by BACTEC MGIT SIRE DST. MIC values were determined using the colorimetric redox indicator (Resazurin/REMA) method, and results were correlated with rpoB gene mutations associated with rifampicin resistance found. Comparing the MIC and critical concentration, we found that 15 of these 101 (14.85%) isolates were misclassified by MGIT-960 as sensitive at standard critical concentration (1.0 mu g/mL) though these were found to have low-level RIF resistance by CRI assay (MIC 0.50 to 1.0 mu g/mL) and NGS. We found that all 15 isolates contained non-synonymous mutations, the commonest being the Ile572Phe (7, 46.66%), followed by Leu533Pro (3, 20.0%), His526Leu (2, 13.33%), His526Asn+Ile572 Phe (1), Asp516Tyr (1), and Leu533Pro+Pro564 Arg (1). These mutations are reported to confer low-level RIF resistance. But we did not find any mutation at MIC <= 0.25 mu g/mL. We found that a significant number of MTB isolates have phenotypic and genotypic discordance. Taking 1.0 mu g/mL of rifampicin as a critical concentration, isolates from approximately 15% of patients are misidentified as susceptible to rifampicin, even when these strains carry low-level drug resistance-conferring mutations and have the potential to develop clinical multidrug-resistant tuberculosis.