Abstract: Tissue samples of liver and spleen from the Muscovy ducks that were suspected to have died from the duck plague were collected for this study.Viruses were isolated from the Muscovy duck embryo fibroblasts (MDEF) for a preliminary identification using hemagglutination assay(HA), immunofluorescence analysis(IFA), qPCR, PCR product sequencing, and animal infection test. Four virus strains, DPVfj1, DPVfj2, DPVfj3 and DPVfj4, were thus identified for further investigation. Subsequently, it was found that these strains (a) would not cause red blood cell agglutination on pigeon erythrocytes; (b) were tested negative on IFA with goose parvovirus, Muscovy duck parvovirus, duck reovirus, duck paramyxovirus, and Tembusu virus; (c) showed positive on test with fluorescence RT-PCR kit; (d) had a positive result on their PCR-amplified pair of primers from the specific fragment of gJ protein gene, JQ673560, and a homologygreater than 99% on the sequence of the fragment with that of the duck plague virus(DPV) gJ protein gene; (e)infected 30-day-old ducks by injection and 5-day-old ducklings by co-habitation showing exactly the same clinical symptoms and postmortem signsas in the natural cases; and, (f)were recovered from the diseased birds. The results seemed to verify the fact that DPV caused the duck plaque in natural environment, and that the isolated strains possessed the virulent nature in question.