Mycobacterium tuberculosis (MTB), resistant to both isoniazid (INH) and rifampicin (RIF) and leading to multidrug-resistant tuberculosis (MDR-TB) can be successfully identified by an 8-week long phenotypic method. However, there is a need for faster diagnostic techniques.In the present study, we evaluated the use of PCR followed by sequencing as a rapid diagnostic tool and determined the incidence of rpoB and katG resistance-associated mutations in Mtb isolates in and around Mumbai. Fifty-Five sputum samples from clinically suspected tuberculosis patients were decontaminated and screened for MTB using Ziehl Neelsen (ZN) staining and Lowenstein–Jensen (LJ) culturing. Extracted DNA from positive samples was subjected to conventional PCR for rpoB and katG genes. In addition, Drug Susceptibility Testing (DST) of isolates was performed by the standard proportional method. PCR products of selected samples of either type i.e., resistant and susceptible by both techniques were sent for sequencing. Fifteen samples (27.27%) were found to be resistant to both drugs phenotypically by DST and by PCR    for rpoB and katG genes. Sequencing analysis revealed point mutations Ser 531 Leu for rpoB gene and Ser 315 Thr for katG gene. The results obtained by direct PCR followed by sequencing method were in accordance with phenotypic method (DST) with high sensitivity and specificity for both genes. The results suggest that the use of molecular techniques can be more suitable for rapid diagnosis of multi-drug resistance in clinical isolates of MDR-TB.