Gallibacteriumanatisis one of the most important microorganisms causing peritonitis in layer chickens. The present study was planned for bacteriological examination of G. anatisand characterization the molecular bases of virulence and antimicrobial resistance of isolated strains. In the current study a total of 50 egg laying chickens were collected from different chicken farms at AL Gharbia governorate, at the period of (2016-2017) samples from (trachea, ovary and oviduct swab) were taken and cultivated on Columbia blood agar, Sheep blood agar, MacConky\'s agarand Dextrose starch agar media for isolation of G.anatis. The results revealed that 8 (16%) of samples were positive for G. anatis isolation. Furthermore, the isolates were subjected to PCR for confirmation, where only 4(8%) of isolates were positive for 16S rRNA23S rRNA. All isolates were sensitive to gentamicin,cefotaxime and ciprofloxacin while, they were resistant to oxytetracyclin and doxycycline which could be explained by the presence of (100%)tetH gene in all tested isolates. Amplification of ( gtxA (rtxA),gyrB and fifA) genes revealed (100%) detection ofgtxA and gyrB genes, while none detection of fifA gene in all tested strains. Consequently, PCR is more specific and it is considered rapid and confirmative type of identification and further studies needed to prepare vaccine against it to overcome the disease and drug resistance.