Infections with Escherichia coli (E. coli) affect poultry of all ages and types. It is frequently associated with poor hygiene, unmet technology needs, or respiratory problems.  The current study was intended to detect the isolation and identification of E. coli as a secondary infection in broiler chickens co-infected with AMPV in Duhok, Iraq. The duration of the study started from September 2024 to March 2025, about 120 nasal and tracheal swabs were collected from infected broilers aged 3-6 weeks from 20 flocks in Duhok city. Furthermore, microbiological culturing and biochemical examinations were done to identify E. coli. The samples were isolated and identified to detect E. coli using MacConckey Agar (MCA), Eosin Methylene blue agar (EMBA), Gram staining, indole test, Methyl Red, Voges Proskauer (MR-VP), citrate, and triple sugar iron agar (TSIA), and antibiotic susceptibility test. Overall (83.3%) of broilers had been infected with  E. coli .Out of 100 isolates were examined for the antimicrobial susceptibility test, It was stated that the isolates exhibited antibiotic resistance Amoxicillin, Cephalothin, Tetracyclin, Fosfomycin, Taichamphnicol, Spiramycin, lincomycin, tilocin, doxacillin were 100 % and ciprofloxacin was 78% and neomycin  was 96% , Cephalexin was 92% and Erythromycin was 98%, Tylosin was 96%, Levofloxacin 54% Trimethoprime-Sulfamethoxazole was 90% , Azithromycin was 80% ,Gentamycin               was 46%  and  Enrofloxacin was 46%. It is essential to use effective prevention and control strategies to decrease the disease.