TB has been announced as a global emerg~ncy of this millennium. It is one of the leading causes of death among adults due to a single infectious agent. Pakistan is sixth among the twenty two Eastern Mediterranean Region countries with the highest burden of disease which is approximately 181 per 100,000. The emergence of drug resistant MTB poses a serious threat to the ongoing efforts to control the disease epidemic. Drug resistance to the first line drugs such as INH and RIF needs to be investigated. In this respect the role of various genes conferring resistance should be studied to find better treatment alternatives. The current practice of drug sensitivity testing requiring approximately three weeks (total turnaround time for MTB culture and sensitivity is around 90 days) is time consuming and a major cause of treatment delay. In this research sputum samples were collected in wide mouth transparent containers from suspected TB patients. After decontamination samples were inoculated onto LJ medium. The colonies grown on the slopes were identified as MTB by standard biochemical test. Isolates were tested on LJ medium for in vitro DST (Drug Sensitivity Testing). MDR was described as resistance to INH and RIF with or without resistance to other drugs. DNA was extracted from the grown samples using kit method. After extraction of DNA, the region from base 2714 to 3232 of katG gene was amplified through peR and the amplified products were sequenced. Analysis of the DNA sequences and mutations was done with the help of BLAST - alignment software. A total of 24 MDR MTB samples were sequenced. Sequence analysis revealed the reported mutation Ser - Thr in katG codon 315 in five samples (21 percent of the total sample size). In this study, an authentic molecular analysis (test) was developed and validated for identification of INH resistant strains in Pakistani population. By studying genetic mutations in katG gene and its association With INH resistance, an alternative can be provided whereby specimens can be tested for these mutations and timely decisions taken. This will not only save the patients from unnecessary treatment delays but will also prevent the administration of drugs to which MTB is resistant and in the long run decrease drug resistance and disease burden.