Using ITS2 gene sequences, the validity of the tapeworm Diphyllobothrium pacificum (Nybelin, 1931), infecting humans on the Pacific coast of South America and in Japan, was assessed. ITS2 sequences of this cestode differed markedly (sequence similarity 79.0-80.2%) from those of the most common human-infecting cestode, the broad fish tapeworm Diphyllobothrium latum (L.), as well as other four species of Diphyllobothrium, including potential human parasites (D. cordatum, D. dendriticum, and D. lanceolatum) and two species of Spirometra (sequence similarity 77.5-81.9%). Interspecific sequence similarity between all but one (D. pacificum) species was 86.1-99.6%, whereas individual isolates of D. dendriticum and D. ditremum exhibited intraspecific sequence similarity of 97.0-98.0% and 98.2-99.9%, respectively. Phylogenetic trees constructed from ITS2 sequences show a markedly distant position of D. pacificum from other species analyzed and also indicate the possible paraphyly of Spirometra. Copyright © 2006 by The American Society of Tropical Medicine and Hygiene.