Escherichia coli O157 is a leading cause of subclinical mastitis in dairy cows and implicated into antimicrobial resistance. So, this study aimed to isolation and phenotypic characterization E. coli O157 from apparently normal cow, s milk and investigate their antibiotic resistance patterns and genes related to this resistance using polymerase chain reaction (PCR). The biochemical evaluation showed that the isolates were positive for Indole, Methyle red, Nitrate reduction, Lactose, and Arabinose sugar fermentation tests, while Voges Proskuaer, Citrate utilization, Urease, and H2S tests were negative and these results confirmed using VITEK® 2 compact. Most of the examined samples were negative for E. coli 122 (81%), while only 28 (19%) of the apparently normal milk samples were contaminated with E. coli. The serological identification revealed that out of 28 E. coli strains, 8 strains were containing O157. The antibiotic susceptibility test indicated that the E. coli strains were highly resistant to gentamycin, ciprofloxacin and tetracycline (100%), ampicillin, cephalothin, kanamycin, nalidic acid and norfloxacin (87.5%), Trimethoprim/sulfamethoxazole (75%) while Erythromycin was (50%). Furthermore, molecular characterization of blaTEM (Beta-lactams) gene (516bp) revealed that 7 of isolates of E. coli O157 strains carried Beta-lactams gene (516bp) while, one isolate was negative. While all isolates were contained TetO (Tetracyclines) and Aada1 (Aminoglycosides) genes that identified at 559bp and 484bp, respectively.