Abstract: In 2018, a severe disease characterized by serious and shaped ulcers in the skins were broke out in the main culture area of yellow catfish (Pelteobagrus fulvidraco) in Zhejiang Province. In order to elucidate the characterization of the causative agent and analyze virulence factors and antimicrobial susceptibility of the pathogen, several bacterial strains were isolated from the lesions and visceral organs of diseased fish. These isolates were subjected to physiology and biochemistry characteristics analyses by using bacterial biochemical identification instrument and biochemical identification micro-tubes. For molecular identification, the 16S rRNA and pyrH were amplified by PCR and compared with sequences deposited in databases, and molecular phylogenetic trees were constructed. Based on the morphological characteristics, phenotypic characteristics, 16S rRNA and pyrH gene sequence analysis, the 11 predominant bacterial strains were identified as Vibrio mimicus. Challenge trials were conducted by intraperitoneal injection, and the results showed that the two representative isolates exhibited high virulence in yellow catfish, skin ulcers occurred in both changed group. The potential virulence of the V. mimicus isolates were examined by phenotypic and PCR assays. All the studied strains exhibited hemolytic activity, and were positive for extracellular protease and gelatinase and DNA degrading activity, but negative for lecithinase activity. Siderophore activity were observed in all V. mimicus isolates. The vmh, ompU and toxR genes were found in all the isolated V. mimicus, whereas none possessed the tcpA, ctxA, st, zot, ace or tdh gene. Antimicrobial susceptibility of the isolated V. mimicus were determined by trace double dilution method and the results showed that the isolates were resistant to ampicillin, sulfadiazine, sulfamethoxazole, sulfamonomethoxine, were sensitive to ciprofloxacin, enrofloxacin, florfenicol, doxycycline, oxytetracycline, thiamphenicol, erythromycin thiocyanate, josamycin, sulfamethoxine, while resistance to neomycin sulphate and thiamphenicol were found in two isolates. Therefore, our study demonstrated the virulence of V. mimicus in yellow catfish as the causative agent of the enzootic. The isolated V. mimicus exhibited the same phenotypic traits and virulence-related genotype, also the biochemical characteristics and antibiotic resistance profile were similar, indicating that they had the same genetic background or source of infection.