Abstract: In order to understand the pathogens of infection in suspected cases of duck viral hepatitis occurring in two farms in Guangdong Province, this experiment was conducted to identify the virus using RT-PCR, virus isolation and sequence analysis of two diseased materials sent from two farms. The virus was isolated from 10-day-old SPF duck embryos and 9-day-old SPF chicken embryos by inoculation of the allantoic cavity after treatment of the disease material, and the whole genome of the virus and VP1 gene were amplified, and the homology alignment, amino acid sequence alignment and phylogenetic tree construction were carried out by using DNAStar 7.0 and MEGA X according to the sequences of the isolates and related virus strains published in GenBank. The results showed that two strains of Duck hepatitis A virus type 3（DHAV-3） were successfully isolated and identified from the disease material, and were named GD18 and GD21. Both materials after challenge did not cause mortality and obvious lesions in chicken embryos, but caused stunting in duck embryos; and the isolate GD21 caused mortality in duck embryos, with characteristic lesions such as generalized hemorrhage and congestion in dead embryos. The two DHAV-3 isolates belonged to the GⅠ genotype and were distantly related to the previously prevalent strains FS, GD and C-GY in Guangdong Province, while they were more closely related to the out-of-province strains SD, JS, CH and EY. Aligned with the amino acid sequences of the reference strains, the isolate GD18 showed two new mutant sites at positions 49 and 67, while the isolate GD21 showed no new mutant site. The results suggested that there might be an epidemic of new strains of DHAV-3 introduced due to the introduction or trade process in duck populations in Guangdong Province.