The presence of koi herpesvirus (KHV) was confirmed in three ornamental koi broodstocks for breeding purposes. The broodstocks showed poor appetite, lethargy, abnormal swimming behavior with gasping movements in shallow water and increased mucus secretion. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells and diffuse lymphocytic–monocytic interstitial nephritis with necrosis of the tubular epithelial cells predominantly found in these three broodstocks. The presence of viral intranuclear inclusion bodies were also observed. KHV was not isolated using KF-1 cell line but PCR positive results at 484bp DNA product was confirmed in all organs of the fish and quatification of KHV genomic DNA using real-time Taqman PCR in all organs ranged from 1.32×10−9 to 1.39×10−9 copy numbers. The analysis of sequencing data of 484bp fragments of KHV DNA of three Korean isolates revealed 100% identities with each other and closely related (98 to 99%) to the known CyHV-3 type of KHV. This report confirmed the presence of KHV infection in one koi commercial farm in South Korea using pathological and molecular methods.