The systematics of lampreys was investigated using complete mitochondrial cytochrome b sequences from all genera and nearly all recognized species. The families Geotriidae and Petromyzontidae are monophyletic, but the family Mordaciidae was resolved as two divergent lineages at the base of the tree. Within Petromyzontidae, the nonparasitic Lethenteron sp. S and Okkelbergia aepyptera were recognized as distinct lineages, Lethenteron morii and Lampetra zanandreai were moved to new genera, a sister species relationship was recovered between Caspiomyzon wagneri and Eudontomyzon hellenicus, and a clade was recovered inclusive of Entosphenus hubbsi and western North American Lampetra (L. ayresii and L. richardsoni). The placement of E. hellenicus as the sister species to C. wagneri reduces the number of genera comprised entirely of parasitic species to two, Geotria and Petromyzon. The recognition of distinct lineages for O. aepyptera and Lethenteron sp. S recognizes, for the first time, lineages comprised entirely of nonparasitic species. Apart from the results mentioned above, monophyly was supported for the multispecific genera Entosphenus, Eudontomyzon, Ichthyomyzon, Lampetra (restricted to European species), and Lethenteron. Intergeneric relationships within Petromyzontidae were poorly resolved, but separate clades inclusive of Entosphenus and Tetrapleurodon (subfamily Entospheninae) and one comprised of Eudontomyzon, Lampetra, and Okkelbergia were recovered.