Besides its zoonotic importance, avian pathogenic Escherichia coli (APEC) causes substantial financial losses in the poultry industry globally. The progress of antimicrobial resistance in APEC is mainly associated with excessive antimicrobial use and improper sanitation. Since its beginning in the 1970s, the VITEK system has developed into the VITEK 2 system, which has used an automated system to perform all the steps required for microbial identification and antibiotic susceptibility rapidly and accurately. The present study aimed to update the available circulating data about APEC isolates by phenotypic identification, sero-grouping of APEC from broilers chickens and breeders in five governates of Egypt, investigation of their antibiotic resistance pattern by VITEK 2 system, and molecular identification of their virulence determinants. The prevalence of APEC isolated from the different internal organs (liver, lung, heart, heart blood, and spleen) was 67.5%. The most prevalent serotypes were O125, 0114, O44, O127, O142, and O78. Virulence-associated genes (iutA, fimC, and papC) were detected at rates of 84.4%, 74%, and 54.8%, respectively. The highest resistance was found against ampicillin (100%), trimethoprim-sulfamethoxazole (80%), and ampicillin-sulbactam (78.5%), which indicates that the poultry farms need a surveillance and intervention system with proper accuracy and rapidity to prevent the misuse of antibiotics and APEC outbreaks.