Phylogenetic relationships between Bothriocephalus species from freshwater and marine teleosts from different geographical regions were studied using internal transcribed spacer–2 and partial 18S ribosomal DNA. The analyses revealed a paraphyly of Bothriocephalus with respect to the genera Polyonchobothrium, Anantrum, and Clestobothrium. The freshwater species Bothriocephalus claviceps, B. acheilognathi, and Bothriocephalus sp. from Dorosoma petenense formed a well-supported monophyletic cluster, with Polyonchobothrium at its base. In contrast, the type species, B. scorpii, clustered within a distinct lineage formed by a heterogeneous assemblage of marine species, Clestobothrium crassiceps and Anantrum tortum, and the freshwater species B. cf. japonicus. This shows that the current morphology-based classification is unlikely to reflect the phylogenetic relationships within this group and will require a thorough revision.