Multi Drug Resistant Tuberculosis (MDR-TB) is an emerging problem of great importance to public health worldwide. Resistance to Rifampicin (RIF) and Isoniazid (INH) are considered as surrogate markers for MDR-TB. Resistance in Mycobacterium tuberculosis to Rifampicin is reported to be conferred by mutation in an 81bp rifampicin resistance determining region (RRDR) of the β subunit of RNA polymerase encoded by rpoB gene corresponding to codons 507-533. Resistance to Isoniazid is conferred by mutation in Catalase-peroxidase gene (katG), alkyl hydroperoxidase gene (aphC) and enoyl acyl reductase gene (inhA). The aim of this study was to identify the mutation conferring resistance to INH and RIF by automated DNA sequencing. Seventy-four isolates of M. tuberculosis were tested phenotypically with four anti tuberculosis drugs namely Rifmapicin (RIF), Isoniazid (INH), Ethambutol (EMB) and Streptomycin(SM) by absolute concentration method. Three isolates from pulmonary tuberculosis (PTB) patients were resistant to RIF with minimum inhibitory concentration (MIC) greater than or equal to 128µg/ml and to INH with MIC greater than are equal to 5µg/ml. The genome of these three MDR-TB isolates were amplified by Polymerase Chain Reaction (PCR) and DNA sequencing was performed. All three isolates showed a point mutation at codon 315 (Ser ® Thr) in the sequenced katG region for INH resistance, but did not show any mutation in the 81bp hotspot sequenced rpoB region for RIF resistance and promoter region of inhA and aphC for INH resistance. Two of three isolates were from patients who had not taken treatment previously which is of great concern with respect to public health.