An unknown disease causing 100% mortality occurred in a koi(Cyprinus carpio)population cultured in Guangdong Province, in April 2013.The moribund koi showed lethargy and unusual swimming behavior such as floating on the pond surface until death.The diseased koi had a mean body length of 25 cm with typical clinical signs including enophthalmia(sunken eyes)and haemorrhages on the skin and base of the fins, and fin erosion.The postmortem examination revealed that liver, spleen, kidney were obviously swollen.No pathogenic bacterium was isolated from internal organs.Positive results were obtained from all detected koi according to a standard PCR diagnosis of CyHV-3 proposed by Office International des Epizooties(OIE).Comparative analysis of nucleotide sequences was performed with the GenBank databases using Blast database network service.The results showed that the putative gene products from infected koi shared 99% identity to that of reported KHV.After filtration treatment, the tissue suspension was injected intraperitoneally into 20 koi.Similar signs appeared and all koi died within 7 days post-infection(p.i.).After filtration treatment, the gills and kidney tissue suspension was inoculated to the CCB monolayer cells.The stable cytopathic effect(CPE)was formed after five blind passages in CCB cells, characterized by severe vacuolation in the infected CCB cells.Under electron microscope(EM), icosahedral viral particles were observed in the cell.The virus particles had an average diameter of 100 nm, and the shape was highly similar to that of KHV.After indirect immunofluorescent antibody test(IFAT), specific fluorescence could be observed on the cell.Based on neighbor-joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result indicated that the pathogen in our study was Asian genotype isolate which was named tentatively KHV-GZ1301.The present study provided important material for virus genesis, evolution, classification, and disease control.