The average mortality of a flock of Hy-Line variety brown hens in Jiangsu Province, China, was 0.44% over 20weeks.Post-mortem inspection revealed that the Pathological changes was consistent with hepatitis-splenomegaly syndrome, including hepatomegaly, serosanguineous peritoneal fluid and hepatic subcapsular haemorrhages.Microscopic lesions of the liver were characterized by multifocal necrotizing hepatitis and mild lymphocytic infiltration.Amounts of avian HEV RNA samples were detected from 56%to 80% of the liver from clinical onset chickens (14/25) and clinical healthy chickens (4/5) by polymerase chain reaction (PCR).Sequencing analysis of a 242-base-pair gene fragment from clinical onset chicken and clinical healthy chicken showed 97.9% to99.2% nucleotide sequence identity with avian HEV genomes, whereas identities ranged from 77.3% to 97.9% compared with other representative sequences.In addition, 40% to 48% of liver samples from clinical healthy chickens (2/5) and clinical onset chickens(12/25) were detectable amounts of MDV meq DNA by PCR, respactively.Sequencing analysis of a 396-base-pair gene fragment showed that the MDV strain belonged to serotype 1 strains.The co-infection rate of avian HEV and MDV was 30% (9/30).In contrast to other reported avian HEV outbreaks that occurred in broiler breeder in China, in the present investigation clinical avian HEV was identified in Hy-Line variety brown hens.This study first found and reported the mixed infection of MDV and avian HEV in chicken.