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Background&Objective: Vibrio species are oxidase positive, gram negative bacilli that predominantly reside in surface waters such as lakes , rivers. They cause predominantly intestinal diseases as well as a few extra-intestinal complications. Vibrio-related diseases often rise during natural disasters such as floods. Vibrio cholerae cause cholera in humans. In this study, the occurance of Vibrio cholerae in the surface waters of Golestan province, was investigated. Materials&Methods: The APW and TCBS agar culture media were used for primary isolation of Vibrio cholerae and the exact species identification were done by performing the following tests oxidase reaction, growth in 0% , 1% , 3% , 6% salt solution, lysine and ornithine decarboxylase, Arginine dehydrolase, ONPG and VP test, simmon citrate, bile esculin, indole, CAMP reaction, string test and specific antisera to V.cholerae 01. to confirm the findings, the special antiserum Ogawa and Inaba, were used. Results: We were able to isolate 42 Vibrio spp. from a total of 54 water samples collected. The species included 35 non-01 V.cholerae (84.2%), 2 V.mimicus (2.63%) and 5 V.cholerae 01 (13.1%) isolat. Conclusion: This study confirmed the existence of Vibrio cholerae 01 in 9% of samples from surface waters of Golestan province.