Gallibacterium anatis (G. anatis) is a member of the family pasteurellaceae which present normally in the reproductive and respiratory tracts of poultry. it causes oophoritis, peritonitis, lowered egg production, and salpingitis, in addition to high mortalities in layers. The purpose of the current work was to detect the prevalence of G. anatis in layer chickens and antimicrobial sensitivity testing of the recovered isolates. A total of 400 samples (100 from cloacal swabs, 100 from tracheal swabs, 100 from lung, 100 from ovary and oviduct) were collected randomly from private commercial layers flocks with high mortality at El-Sharkia Governate, Egypt. Collected samples were subjected for distinct bacterial examination for identification of G. anatis bacteria. Recovered isolates were tested for antimicrobial sensitivity testing and detection of the prevalence of multidrug-resistant (MDR) strains. From 400 diseased examined samples, 120 were positive for G. anatis as follows (cloacal swabs 27/100; 27%, tracheal swabs 35/100; 35%, lung 35/100; 35% and ovary and oviduct 23/100; 23%) of layer chickens. The overall incidence of G. anatis was 30% in the recovered samples.  Recovered isolates were highly resistant to doxycycline, amoxicillin, and gentamycin with 98%, 96%, and 95%, respectively. All isolates were MDR. Isolates were sensitive to florfenicol (90%), erythromycin (96%), difloxacin (44%), and sulfamethoxazole-trimethoprim (57%). The current study revealed the serious and wide prevalence of multi-drug resistance of G. anatis in layers. Retrieved isolates were highly resistant to amoxicillin, doxycycline, and gentamycin which make a serious problem in the poultry industry with dangerously public health threat.