This paper studied the identification, drug resistance and the molecular resistance mechanism of five Vibrio strains isolated from diseased Cynoglossus semilaevis. The five strains were identified as V. cyclotrophicus by a biochemical test combined with a molecular method (16S rRNA, HSP60 and mreB genes). The broth dilution method was used to determine the antimicrobial susceptibility of the strains to 14 antimicrobials. The PCR amplification and DNA sequencing were used to detect antimicrobial resistance genes, integrons and the gene cassettes. The results indicated that the five strains were resistant to ciprofloxacin, streptomycin, erythromycin, florfenicol, ampicillin, SMZ, TMP/SMZ and rifampicin, and carried qnr VC, bla_CARB-17, strA, strB, sul genes. Class I intergron, qacEΔ1 and sul1 genes were present in JS291 and JS295 strains. The integron-gene cassettes of JS291 were encoding dihydrofolatereductase (dfr16), aminoglycoside-(3″)(9)-adenylyltransferase (aadA2), and the cassettes of JS295 were encoding rifampin ADP-ribosylating transferase (arr-3), dihydrofolate reductase (dfrA27), respectively. These findings suggested that the five strains exhibited multi-drug resistance and the multi-drug resistance phenotype may be closely related to the antimicrobial resistance genes.