The pathogenicity of Pseudomonas aeruginosa bacteria is primarily due to the production of extracellular enzymes and the expression of virulence factors, such as toxA and oprL, is involved in the pathogenic process. The use of PCR for the identification of these genes can help in the early detection and control of infections caused by this bacterium. In this study, a total of 480 samples were gathered from various internal organs (Isolated Pseudomonas aeruginosa; liver n=85, lung n=97, gall bladder n=100, and kidney n=87) of broiler chickens of different ages across five administrative centers in Fayoum governorate and then bacteriologically examined for reporting the prevalence rate of P. aeruginosa. The result confirmed that 327 out of the 480 samples were positive for P. aeruginosa, with a prevalence of 77.5%, of which the highest isolation rate was from Tamyyah (91.6%), followed by Ibshaway (85%), Itsa (76.8%), and Sinnuris (52%).Furthermore, determining the virulence gene profiles of eight selected isolates using polymerase chain reaction (PCR) indicated that all eight tested isolates (100%) were positive for the harbored oprL gene, whereas only five P. aeruginosa isolates (62.5%) were positive for the toxA gene. Based on the obtained results, it was concluded that the widespread occurrence of this opportunistic pathogen in poultry production systems is particularly concerning, as it suggests that P. aeruginosa has become a major concern for poultry health and food safety in the studied regions.