Pasteurella multocida has gained several mechanisms that developed antibiotic resistance due to the misuse and abuse of antibiotics. Deep screening of bacterial molecular mechanisms and virulence factors will help find new threats for animal and human diseases. Therefore, this research aims to isolate and identify P. multocida from various affected Egyptian farms of layers and breeders and study phenotypic characterization, antimicrobial sensitivity, and PCR genotypic identification. Finally, the pathogenicity of isolated P. multocida strains was evaluated in SPF chickens. A total of 312 samples, from internal organs, were subjected to bacterial isolation. The isolated P. multocida strains were identified biochemically using API20NE and genetically by P. multocida-specificPCR. The antibacterial effect of some antibiotics was determined on the basis of the Clinical and Laboratory Standards Institute. In the pathogenicity test, the challenge dose contains 2.9x108 CFU/0.5mL via the IM route. The phenotypic and genotypic characterization revealed 47.7%(149 samples) positive samples for P. multocida. Genetic characterization confirmed that 5 out of 8 isolated strains were P. multocida type A by PCR. In the sensitivity test, the isolates were susceptible to ciprofloxacin and levofloxacin and resistant to clindamycin and erythromycin. Highly pathogenic strains inoculated into SPF chickens resulted in 84% (21 chickens from 25) morbidity with severe clinical signs. The results of this study indicate that P. multocida can potentially cause significant losses to flocks of layer and breeder chickens in Egypt due to being a contagious pathogen. The isolated strains were highly pathogenic to SPF chickens.