Abstract: Objective To analyze the virulence genes and genotyping characteristics of environmental O1/O139 Vibrio cholerae(V.cholerae)in Guangdong province and to provide the basis for the prevention and control of cholera.Methods Eight pairs of primers were designed according to ctxA,ace,zot,tcpA,tcpI,hlyA,ompU,and toxR.The multi-plex PCR(MPCR)was established to detect 90 V.cholerae O1/O139 strains isolated between 2008 and 2009(69 aquatic strains,16 sea food strains and 5 clinical strains).Genotypes associated with the virulence were determined then accord-ing to the result of the MPCR.Results The hlyA and toxR genes were positive in all the isolates.Three of five clinical isolates were detected for eight virulence genes and the other two isolates displayed the genotype of virulence with toxR⁺,ompU⁺,zot⁺,tcpA⁺,tcpI⁺,hlyA⁺,toxR⁺,and tcpA⁺.For the aquatic isolates,14 Inaba strains(34.15%,14/41)were hlyA⁺,toxR⁺,ompU⁺,ace⁺,zot⁺,tcpI⁺,while 12 Ogawa strains(66.67%,12/18)and 7 O139 strains(70%,7/10)were hlyA⁺and toxR⁺.For sea-food isolates,3 Inaba strains(75%,3/4)were hlyA⁺,toxR⁺,ompU⁺,and tcpI⁺; while the Ogawa strains presented different genotypes.Conclusion The environmental O1/O139 Vibrio cholerae in Guangdong province display widespread non-toxigenic strains with virulence genotype diversity.