The mechanisms of resistance to levofloxacin for Aeromonas hydrophila isolated from diseased fish and selected in vitro were examined in this study. Levofloxacin-resistant mutants were obtained by selection of A. hydrophila in vitro. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were sequenced in Lev(R) strains and reverse mutation strains. All Lev(R) strains carried a point mutation at codon 83 (Ser → Ile), and one strain (25 %) harbored a mutation at position 92 (Leu → Met) in the GyrA-QRDR. After being transferred in a levofloxacin-free medium, one strain of the mutants was successfully reversed and the reversion was related with mutations of GyrA-QRDR at positions 81 (Gly → Asp) and 83 (Ile → Ser). No amino acid alteration was found in the ParC-QRDR. In addition, the minimum inhibitory concentration (MIC) of levofloxacin for the mutants was lower in the presence of reserpine, and all mutants were also resistant to some of the other quinolones. It was found that the mechanism of levofloxacin resistance of A. hydrophila selected in vitro was related to gyrA of type II topoisomerase, and an efflux mechanism was involved in the resistance as well.