Introduction: Resistance to rifampicin is considered to be a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB). Objective: The present study was undertaken to evaluate the drug resistance profile and the resistance-conferring mutations of MDR-TB species in a tertiary care hospital, Central Kerala. Methods: Clinical specimens (n=404) were subjected to Ziehl-Neelsen (Z-N) staining and culture and drug susceptibility testing (DST) using liquid (BD BACTECTM Micro MGITTM {Mycobacteria Growth Indicator tube). RIF resistant isolates (n=5) detected phenotypically along with randomly selected RIF sensitive isolates (n=5) were randomly selected and rpoB gene of all the amplified strains was sequenced and mutations analysed. Results: Out of 404 samples from clinically suspected cases of M. tuberculosis, Mycobacteria was grown in 48 (11.9%) samples. Amongst the forty-eight M. tuberculosis isolates, 41.6 % were sensitive to all five drugs and 6.2 % were resistant to all five drugs. Of the total of 48 culture-positive TB cases, one (2.08%) was found to be MDR-TB and additional resistance was observed in one (2.08%) isolate. Of the ten isolates (five RIF resistant isolates detected phenotypically along with five RIF sensitive isolates) which was amplified by PCR, M. tuberculosis DNA was not detected by PCR in one of the isolates. Mutations detected occurred in codon 531 with Ser __ampersandsignrarr; Leu substitution. Conclusion: Molecular methods along with conventional detection methods help in understanding the transmission dynamics of tuberculosis and could be used as a tool in controlling the transmission of tuberculosis and thereby achieving the elimination of tuberculosis by 2030.