Mortality of cultured koi Cyprinus carpio occurred up to 25% at a commercial fishery in North Chungcheong Province of South Korea. Gross observations showed that long-white parasites have blocked and filled the intestine, protruding into the mouths of the fish. Histopathology of the damaged intestine was checked and the structure of the parasite was examined under scanning electron microscope (SEM). Finally, V4-18S rRNA gene Polymerase Chain Reaction (PCR) and nucleotide sequencing methods were conducted and confirmed that the parasites were Bothriocephalus acheilognathi. This study provides the basic and practical diagnostic methods for B. acheilognathi infection in cultured koi.