M. tuberculosis strains were isolated from clinically and bacteriologically confirmed patients to evaluate the susceptibility of clinical M. tuberculosis isolates to fluoroquinolone and to obtain molecular epidemiological information in Zambia,. The pathogens were subjected to susceptibility testing with isoniazid, rifampicin, ethambutol and streptomycin. The minimum inhibitory concentrations to ciprofloxacin, sparfloxacin and levofloxacin were also evaluated. The gyrA, fluoroquinolone resistance-determining region (QRDR), was sequenced and analysed. As a result, three of the 16 strains examined were resistant to isoniazid, rifampicin and⁄or streptomycin. All of the strains were susceptible to ciprofloxacin, levofloxacin and sparfloxacin. However, a unique gyrA gene variation of M. tuberculosis was identified in the isolates. One strain had a mutation (T73A) in QRDR. Additionally, 81.25% (13⁄16) of the strains tested had Thr at codon 88. Several variations of gyrA gene have been reported in relation to drug resistance. The gyrA variation data will be useful as epidemiological information. It may be important to monitor fluoroquinolone susceptibility even in developing countries for use against resistant M. tuberculosis infection, even though no fluoroquinolone resistance was observed in this study.