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Objective: Contamination of surface water with pathogenic organism is highly dangerous for people who used it for drinking or for domestic activities. Detection of Escherichia coli in water can be used as a general important indicator of fecal contamination. This study investigated the occurrence of fecal E. coli, two important toxigenic types of E. coli isolates and their antimicrobial resistance in water samples collected from the surface running source of Wadi Shueib in Jordan. Methods: A total of 51 water samples were collected from three different locations of wadi shueib over a three month, July through September, 2016. For each sample, 200 ml of water was collected in sterilized containers. All samples which were positive for fecal E. coli were subcultured on Eosin Methylelne Blue Agar and incubated at 37 oC for 24 hours. E. coli isolates were identified by API20 E test, and all isolates were tested for antimicrobial susceptibility and for the presence of virulence genes of enterotoxigenic E.coli (ETEC) and enterohaemorrhagic E.coli (EHEC). Results: A total of 46/51 (90%) of water samples were contaminated with fecal E. coli. The 46 E. coli isolates were resistant in the range between 4%-76% to commonly used antibiotics in the treatment of infection in Jordan. Multidrug resistant isolates to at least three antibiotics accounted for 17/46 (37%) of the isolates. Out of 46 fecal E. coli isolates, 4 (8.7%) were ETEC and 2 (4.3%) were EHEC as detected using PCR. Conclusion: This study indicated that the surface running water of Wadi Shueib is contaminated with potential enteropathogenic E. coli, Molecular characterization of virulence genes and antibiotic resistance among fecal Escherichia coli isolated from surface water of Wadi Shueib-Jordan Shereen Z. Burjaq1, Saeid M. Abu-Romman2, Moawya A. Haddad3 1 Department of Medical Analysis, Faculty of Science, Al-Balqa' Applied University, Salt, Jordan. 2 Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University Salt, Jordan. 3 Department of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balqa Applied University Salt, Jordan. Contact information: