Abstract: To investigate the cause of sudden death of the koi (Cyprinus carpio), the moribund koi that showed typical symptoms including sunken eyes and head, rotted gill and haemorrhages on the skin were examined. No bacteria were isolated from spleen, liver and kindey of diseased fish by bacteriological examination, but a large number of viral nucleocapsid were found in gill tissue by electron microscopy observation. After filtration treatment, the gills, brain, liver, spleen, and kidney tissue suspension was inoculated to carp fin cells, Cytopathic effects (CPE) were presented 7 days post co-cultivation. In artificial infection test, the death rate of fish by infecting tissue homogenate and culture liquid of infected cell reached 93.3% and 86.7% respectively. Histopathological analysis showed that the target organs included the gill, liver and kidney. Positive results were obtained from diseased koi according to a PCR diagnosis of koi herpesvirus (KHV) proposed by Office International des Epizooties (OIE), and high concentration of KHV was detected in gill and kidney tissue by fluorescence quantitative PCR. Based on neighbor-joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result indicated that the KHV isolated in our study was Asian genotype isolate. Furthermore, according to the sequence alignment of Marker I and Marker II regions of KHV, the KHV detected in this study belongs to I⁺⁺II^–genotype, and was named KHV-TJ1601. The present study demonstrated that the I⁺⁺II^–genotype KHVwas isolated from the naturally infected fish in North China for the first time, and provided important material for virus genesis, evolution, classification, and vaccine preparation.