ABSTRACT   The present study was conducted in Kafrelsheikh governorate to isolate P.aeruginosa from 120 ducks, sampling from cloacal swab, internal organs and tracheal swab of freshly dead, healthy and diseased ducklings at different ages which showed depression, emaciation, ocular and or nasal discharges, diarrhea and sometimes enlargement of hock joint and had lesions of airsacculitis, congested liver, catarrhal enteritis and sometimes presence of gelatinous material in the hock joints with rate of isolation of 16.66% (20 samples were positive). Serological identification of suspected isolatesof P. aeruginosa showed that 16 isolates of polyvalent I group Iand 4 isolates of polyvalent II group J. Susceptibility of the isolatedP. aeruginosa for 11 antibiotics demonstrated that the highest resistance was noted against Erythromycin, Oxytetracycline, Ampicillin and Amoxicillin (95%). The highest sensitivity of P. aeruginosa was observed againstFlorphenicol(80%) and it used for treatment in experimental design and give good results.Multiplex PCR used to identify virulencerepresented bytoxA gene, lasI gene and oprL gene ofP. aeruginosa show that all isolates where positive for the 3 examined virulence genes and these indicate that all isolates were highly virulent strains and antibiotic resistance genesrepresented by qnrS gene, blaCTX gene and mexR gene ofP. aeruginosa showed that all isolates where positive for the 3 examined AB resistanceexept isolate No 4 were negative forblaCTX and these indicate that all isolates were highly AB resistanceexept isolate No 4 wasnot haveblaCTXresistance gene.Experimental infection was done to study the Pathogenicity of P. aeruginosa which isolated from diseased ducklings on newly hatched ducklings with dose of infection of 0.2ml of 1x10⁷ cfu/ml s/c and use the potent antibiotic florphenicol for treatment of experimentally infected ducks. Our study proved that virulence genes owned by the P. aeruginosa confirming its pathogenicity for ducks, especially in the presence of oprL gene which plays a great role in antimicrobial resistance, so biosafety was recommended for hatcheries and farms, the hygiene, cleaning and disinfection will reduce P. aeruginosa spreading in the farms.