This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 --> Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.