TheEnterobacteria harboring the extended-spectrum-β-lactamase (ESBL) are a serious threat to public health particularly in developing countries. The study was aimed to investigate the prevalence and genomic characterization of ESBL producing E. coli from poultry, environment and poultry farm workers in Islamabad and Rawalpindi Pakistan.A total of 250 poultry samples, 92 poultry environmental samples, and 50 poultry farm worker’s urine samples were screened for ESBL producing E. coli using ChromeIDTM ESBL agar. Biochemical confirmation of the isolates was carried out by API 20E. The antimicrobial susceptibility profiling and phenotypic confirmation of ESBL producers were performed as per CLSI 2018 guidelines. The minimum inhibitory concentration of ESBL producing isolates was determined by the broth dilution method. Phenotypically confirmed ESBL producing strains were further subjected to molecular characterization for the presence of ESBL and carbapenemase-producing genes using PCR. Of 392 samples, 219 ESBL positive E. coli were recovered and among these 156/213 (73.2%), 42/63 (66.6%) and 21/27 (77.7%) were from poultry, environmental water, and urine samples, respectively. The PCR results revealed that71.2% of blaCTX-M, 67.5% blaCTX-M -1 and 62.2% blaTEM producing isolates were recovered from poultry, 19.1% blaCTX-M, 21.6% blaCTX-M -1, 36.8% blaTEM from environment and 9.6% blaCTX-M, 10.8% blaCTX-M -1 0.8% blaTEM from urine samples. Moreover, blaCTX-M-2 (n=19), blaCTX-M-8 group (n=19) and blaCTX-M-9 group (n=27) were only observed in poultry samples. In addition, carbapenemase encoding genes as 9.6% (19/219) blaIMP, 21.4% (47/219) blaVIM and 15.5%(34/219) blaNDM were also detected. ESBL producing E. coli exhibited resistance against cephalosporins, β-lactamase inhibitors, monobactam, folate-pathway inhibitors, fluoroquinolones, and aminoglycosides. This study investigates the high prevalence of ESBL producing E. coli in chicken, farmworkers, and water that is alarming and could lead to serious threats to both livestock and public health.