Fluoroquinolones are one of the new classes of antimicrobial agents that are being increasingly used against tuberculosis, the most important ones are ciprofloxacin, ofloxacin, and the new generations drugs-gatifloxacin and moxifloxacin. The clinical isolates of Mycobacterium tuberculosis (47 ofloxacin- susceptible and 71 ofloxacin-resistant) strains obtained from individual patients from various parts of India were analyzed for gyr A mutation in quinolone resistant determining region (QRDR). Most of the mutations were seen clustered in the codons 90, 94 and 95, which is a hot spot region of QRDR. The types of mutations were correlated with the in vitro susceptibility pattern of the strains to ofloxacin. The resistance to fluoroquinolones was observed predominantly due to gyr A mutations. Gyr A being a gene with house keeping functions, is highly conserved in various mycobacterial species. It was adapted for PCR-RFLP assay using gyr A gene to see the difference in the profile of 23 standard species of mycobacteria. M.tuberculosis, M.avium, and M.intracellulare produced a different profile with two different restriction enzymes.