Comparison of the quinolone resistance-determining regions (QRDRs) in 42 Mycoplasma bovis clinical isolates revealed amino acid substitutions at both GyrA (position 83) and ParC (position 84) in 10/11 enrofloxacin-resistant strains. The mutation present in the parC QRDR was discriminative for enrofloxacin resistance by parC PCR-restriction fragment length polymorphism. Comparison of molecular profiles by insertion sequence typing suggests that the currently prevalent enrofloxacin-resistant M. bovis strain evolved by selection under field conditions from one of the susceptible strains.