A. hydrophila is a foodborne pathogen and one of the most important pathogenic bacteria which has a great hazard on public health and fish industry. It causes a variety of diseases in humans and different animal species from aquatic to terrestrial animals. In fish it responsible for fatal aeromonad septicemia which cause significant economic losses to aquaculture worldwide each year. This study was designed to investigate the prevalence, antimicrobial resistance, virulence characteristics and genotyping of A. hydrophila isolates from diseased Nile tilapia (Orechromus niloticus) in aquaculture farms in three Governorates in Egypt. A total of 100 diseased Nile tilapia, were collected from four aquaculture fish farms, located in Sharkia (total n = 50: Al-Hosseinieh (n = 25) and Al-Abbassah districts (n = 25)), Port Said (Sahl- ElTina), and Kafr ElSheikh Governorates (n = 25, each), Egypt. Of the 41 Aeromonas isolates recovered from diseased fish, 39 (39%) were confirmed by 16S rDNA-PCR amplification.Virulence characteristics and antibiotic resistance patterns of 39 A. hydrophila isolated from 100 diseased Nile tilapia from three Governorates in Egypt were investigated. The genetic diversity of A. hydrophila isolates was determined by ERIC-PCR. Almost all isolates were resistant to ampicillin and amoxicillin. The least resistance was noted for doxycycline and gentamicin (7.7%), streptomycin (10.3%), trimethoprim/sulfamethoxazole (17.9%), chloramphenicol (25.6%), colistin (43.6%), and erythromycin (47.7%). All tested isolates were susceptible to ciprofloxacin (100%). MAR index values ranged from greater than 0·2–0·6 among the MDR isolates (87·2%). Of the 39 tested isolates, 16 (41.03%) presented at least one of the virulence genes under study. Thirteen isolates (33.33%) were positive for the aer gene; 9 (23.1%) harbored lip, 2 (5.13%) carried the hlyA, and one isolate (2.56%) was positive for ast gene. Aeromonas hydrophila isolate containing 4 virulence genes (aer/hlyA/ast/lip) induced the highest mortality (76.67%) in inoculated fish followed in descending order by isolates harboring lip and aer genes (66.67%), hlyA (63.3%), aer (60%), and lip (56.6%). The analysis of banding patterns generated by ERIC-PCR allowed differentiating the 20 A. hydrophila isolates from 4 different geographic regions into 3 main clusters, each contains isolates from different localities. This considered as evidence of isolates from the same locality did not cluster close to each other. The fingerprinting profiles of isolates were diverse without any obvious regional or cluster-specific isolates.