Thirty-seven Vibrio cholerae strains were isolated from surface water sources at 5 different locations in Tehran, Iran during 2006 and were identified as non-O1 and non-O139 isolates. PCR for SXT element and class 1 integron was positive for 19% and 5.4% of isolates, respectively. PCR for virulence associated-genes within the vibrio pathogenicity island (VPI) gene cluster showed the presence of LJ, int and RJ in 8, 59 and 30% of the isolates, respectively. None of the V. cholerae isolates contained the toxin encoding genes (ace, zot, ctx) in the CTX genetic element. Biochemical fingerprinting using PhPlate system (PhP-RV) was able to type all strains and resulted in 8 common types (containing 78% of the isolates) and 8 single types (22%). Out of 37 isolates, only 26 isolates were typeable with pulsed-field gel electrophoresis (PFGE) producing banding patterns. The results presented in this study showed no genotyping correlation between the V. cholerae isolated from surface water and the clinical setting which had been reported previously by this laboratory. Furthermore, combination of PFGE and PhP-RV methods was proved beneficial for non-typeable V. cholerae isolates.