Abstract: Three virus strains were isolated from the diseased layers that experienced abrupt reduction in egg-laying in different regions.These isolated viruses could cause SPF chick-embryo and Muscovy duck-embryo to die with hemorrhage in embryo-livers.Healthy egg-producing layers inoculated with these isolates showed symptoms similar to that of the field cases,and the same viruses could be reisolated.These isolates could proliferate in MDEF causing cytopathic effect.They showed no haemagglutination activity,were sensitive to chloroform and ether,and resisted to FUDR.PCR and IFA screening indicated that the isolates were negative to avian fluvirus,Newcastle Disease Virus,egg-laying-reduction syndrome virus,infectious bronchitis virus,infectious laryngotracheitis virus,duck reovirus,duck parvovirus,infectious bursal disease virus and goose parvovirus;but positive to flavivirus.The nucleotide sequence from the isolates had greatly homogeneity with duck flavivirus isolates.These results indicated that the isolates were the viruses causing the layer′s reduction in egg-laying in recent years,and that they belonged to the genus,Flavirirus.