Listeria monocytogenes is the causative agent of listeriosis and food-borne disease that can lead to meningitis and bacteremia. L. monocytogenes is the most pathogenic species in the genus Listeria. A total of 400 random samples of muscles, liver, spleen and kidneys (100 samples each) were taken from 100 diseased chickens. All samples were obtained from different poultry farms and markets in El- Gharbia Governorate to estimate the prevalence of Listeria monocytogenes in chicken. The bacteriological examination of the samples resulted; 53(13.25%) isolates as 11, 21, 8 and 13 from muscles, liver, spleen and kidneys respectively. The antimicrobial sensitivity test showed that the isolated L. monocytogenes were sensitive to sulfamethoxate-trimethoprim (91.6%) followed by ampicillin, gentamycin, vancomycin and chloromphenicol (83.3%) followed by ciprofloxacin (66.6%) followed by erythromycin and tetracycline (50.0%), while the isolated strains were completely resistant to cephalothin. All L. monocytogenes strains were virulent where all of them were positive to CAMP test and Anton's test. The PCR results for six studied strains of isolated L. monocytogenes showed that all genes (16S rRNA; inlA; prfA and hlyA) were detected (100.0%) while plcA gene was not detected.