Duck hepatitis A virus (DHAV) is the cause of duck viral hepatitis, which is an acute and infectious illness with a high mortality rate. In this study Thirty samples from several duck farms in Qalyubia Governorate were used in this investigation between 2023 and 2024. The tested birds which ranged in age from 7 to 12 days had neurotic symptoms including ataxia, opisthotonos, spasmodic kicking, swift death and enlarged liver showing noticeable petechiae hemorrhages. Using allantoic sac isolation and molecular characterization by RT-PCR, these samples were utilized to identify DVH in SPF-ECEs. After the eggs were harvested, the embryos displayed bleeding, stunting, and greenish necrosis of their livers. 250 pb 5UTR gene fragment was amplified by RT-PCR, and 880 pb VP1 gene was amplified by subtyping, indicating that the isolate is related to DHAV-3. The affirmative subtyped sample was next subjected to sequencing analysis and phylogenetically examined. The examined strain's VP1 nucleotide sequence revealed differences with Chinese and Korean-Vietnamese strains (86.5% to 88.1%) as well as 66.8% and 96.8% to 100% similarity to vaccinal and Egyptian strains, respectively. The pathogenicity of the isolated strain was then assessed using two distinct age groups. By monitoring liver and kidney functions and doing a histological study, the results verified that our isolate is a highly pathogenic virus.