Tapeworms of the genus Diphyllobothrium (Cobold, 1858) are widely distributed all around the world and some of them are agents of human diphyllobothriasis. Approximately 50 species have been described within the Diphyllobothrium genus but only 13 are human pathogens. Species identification by using morphological criteria is very difficult. We determined the value of 18S ribosomal RNA gene, internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 gene (COI) sequences to differentiate between Diphyllobothrium isolates. Sequences from 18 isolates (larvae or adults) of D. latum, D. nihonkaiense, D. ditremum, D. dentriticum and D. stemmacephalum species were obtained. COI region sequences analysis was clearly more discriminative than those of the ITS1 and 18S rRNA and was a useful tool for identifying specimens.