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Waterborne outbreaks of diseases caused by pathogenic strains of E. coli have been described in many parts of the world. For the past five years in a row, water has been the main source of contamination. Aleppo River is highly polluted by the sewage water that transferred directly without processing. So, we conducted estimate total count of bacteria, the census total coliform group, also detected E. coli and especially serotype E. coli O157:H7 in water samples that were taken from eight villages passed by the river. Our results showed significant differences between the collected samples, the total count of bacteria and total coliforms in the first site (Handarat) for water samples were the lowest, but in the last site (Jezraya) were the highest among all samples. Molecular characterization was done for eight strains of E. coli using the primer (COL-1) and eight strains of E. coli O157:H7 using the primers (OPA-03, OPA-13 OPC-12, OPE-20) in RAPD. After gel documentation, we have got eight different bands from isolated strains of E. coli, and forty-five total bands with thirteen polymorphic bands from E. coli O157:H7, also showed that primer OPA-13 was the best primer for detect E. coli O157:H7. This article also showed that RAPD analysis had a high discriminatory capacity for typing E. coli isolates. Because of its simplicity and rapidity. © 2019 Author(s).