Severe hydropericardium syndrome (HPS) has been present in layers in the northeast of China since June 2015, with mortality rates varying from 30 to 90%. Dead layers had severe hydropericardium with pericardial volumes of 5 to 20 mL, as well as inclusion body hepatitis. Laboratory investigations led to the isolation of a fowl adenovirus strain, HLJFAd15, from the liver tissue of dead layers. Natural deletions of ORF19 and ORF27 were found in this clinical strain by complete genome sequencing, which was identified with the novel genotype recently prevalent in China. The pathogenicity characterization was conducted in 35-day-old SPF chickens using HLJFAd15 with novel genotype of fowl adenovirus serotype 4 (FAdV-4). The reproduction disease cases of HPS with mortality rates of 76.9% by oral administration and 100% by intramuscular injection were induced successfully by challenging SPF chickens, respectively. Non-enveloped viral particles with a mean diameter of approximately 80 nm were found in the livers of virus-infected SPF chickens. Our study revealed that HLJFAd15 was identified with the novel genotype strains recently emerging in China by complete genome sequencing, and the strain was capable of causing HPS by the pathogenicity analysis. However, although there is currently no commercial vaccine against the novel genotype FAdV-4, the animal infection model established in this study was valuable for vaccine evaluation and development.