The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the A and B subunits of DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in mycobacteria. We looked to see if the assignment of mycobacterial species could be obtained simultaneously by analysis of the corresponding nucleotide sequences. PCR sequencing of gyrA and gyrB QRDRs was performed for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories. Nucleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.