To find the association of gvrA gene mutation with ciprofloxacin resistance in M. tuberculosis, 21 clinical isolates of ofloxacin-resistant M.tuberculosis, determined by absolute concentration method, and 20 ciprofloxacin-susceptible clinical isolates were also included in this study. Resistant clinical isolates selected on media containing ciprofloxacin were tested for ciprofloxacin and ofloxacin susceptibility testing by radiometric method (BACTEC). The results compared well with the absolute concentration method as all isolates were resistant to ciprofloxacin. For the genetic information, gyrA gene of each isolate was amplified and was determined mutation by DNA sequencing and heteroduplex formation analysis. DNA sequencing could detect the gyrA mutation in 18 isolates; Asp Asn (n=6), Asp Ala (n=7), Asp Gly (n=4), Asp Tyr (n=1) and the 3 isolates lacked mutation. No mutation was found in 20 ciprofloxacin-susceptible clinical isolates. Evaluation of polymerase chain reaction-heteroduplex formation (PCR-HDF) analysis for rapid susceptibility testing revealed that this technique could not detect the differentiation between susciptible strain H37Rv and resistant strain.