Aeromonas hydrophila is one of the main opportunistic pathogens in aquaculture. Quinolones are one of the main fishery antibiotics. However, the frequent and unreasonable use of quinolones has led to the emergence of bacterial resistance. The research on the molecular characteristics of quinolone resistant plasmid of Aeromonas spp. is still in the initial stage. In this study, to investigate the relationship between endogenous plasmids and quinolone-resistant phenotype of A. hydrophila isolates, a plasmid termed pAhW39, from a quinolone-resistant A. hydrophila strain W39 which was isolated from a diseased Megalobrama amblycephala from a fish farm in Xiantao, Hubei Province, was cloned and characterized. pAhW39 was 6 739 bp long, had a GC content of 46.13%, and contained four putative open reading frames (ORFs) (nspv, nspV-like, repB and qnrS2). The GC content of pAhW39 was lower than the GC content of the currently published A. hydrophila chromosomal DNAs which ranged from 60.1% to 62.0%, suggesting a possibility that the plasmid pAhW39 might originate from other strains through the horizontal transfer of plasmids. nspV and nspV-like encoded type II endonucleases NspV and NspV-like, respectively. The repB encoded a replication protein RepB and qnrS2 encoded a quinolone resistance protein QnrS2 which usually confers increased resistance to quinolones. pAhW39 was stable in A. hydrophila strain W39 in the absence of selective pressure. Compared with plasmid pAhW39 elimination strain W39-C, the minimum inhibitory concentrations of nalidixic acid and ciprofloxacin against strain W39 were increased 16-fold and 8-fold, respectively, indicating that the plasmid pAhW39 containing qnrS2 might mediate the resistance of strain W39 to quinolones. Comparative genomics analyses revealed that the plasmid pAhW39 had 99.9% DNA sequence identity over its whole length with the plasmid pAER-e58e from Aeromonas sp. strain ASNIH2 isolated from wastewater from a hospital in Maryland, USA. The result implied that plasmid pAhW39 (or pAER-e58e) was widespread and stable, and reminded us that the existence of plasmid-mediated quinolone resistance in Aeromonas spp. in fish might cause a more widespread dissemination of resistance to quinolones in aquaculture. This should attract more attention.