Pseudomonas aeruginosa is an opportunistic pathogen causes serious problems in broiler farms and this pathogen has an economical importance in broiler sector. A total of 200 broiler chickens of different ages [84 broiler chicks (1-7 days) and 116 broiler chickens (1-6 weeks)] were collected from different broilers farms for isolation and identification of P. aeruginosa. Different samples were taken from internal organs (liver, gall bladder, lung, heart blood, intestine, kidney, yolk sacs) as well as cloacal swabs. The samples were examined bacteriologically, 28 isolates (14%) of P. aeruginosa were isolated and identifiedby biochemical reactions as well as API 20E system. The yolk sac and cloacal swabs samples gave the highest recovery rates with percentages of 15.5% and 12.6%, respectively. PCR assay confirmed the existence of P. aeruginosa DNA in ten isolates by using 16S rRNA gene. Also,PCR assay was carried out to detect the presence of virulence genes as oprL, toxA and aprA. The oprL gene was present with a percentage of 100%, toxA was present with a percentage of 80%, and aprA gene with a percentage of 40%. Also, sequencing of 16S rRNA gene of P. aeruginosa was submitted to Gen Bank with accession number MW051028 which was 100% identical to P. aeruginosa strains.