The genus Mesocestoides Vaillant, 1863 includes tapeworms of uncertain phylogenetic affinities and with poorly defined life histories. We previously documented 11 cases of peritoneal cestodiasis in dogs (Canis familiaris L.) in western North America caused by metacestodes of Mesocestoides spp. In the current study, DNA sequences were obtained from metacestodes collected from these dogs (n = 10), as well as proglottids from dogs (n = 3) and coyotes (Canis latrans Say, 1823 [n = 2]), and tetrathyridia representing laboratory isolates of M. corti (n = 3), and these data were analyzed phylogenetically. Two nuclear genetic markers, 18S ribosomal DNA and the second internal-transcribed spacer (ITS 2), were sequenced. Phylogenetic analysis of the 18S rDNA data recovered a monophyletic group composed of all samples of Mesocestoides spp., distinct from closely related outgroup taxa (Amurotaenia Akhmerov, 1941 and Tetrabothrius Rudolphi, 1819). Initial analysis of the ITS 2 data resolved 3 clades within Mesocestoides. Two proglottids from dogs formed a basal clade, a second clade was represented by tetrathyridial isolates, and a third clade included all other samples. Interpretation of these data from an apomorphy-based perspective identified 6 evolutionary lineages. We also assessed whether metacestodes from dogs (n = 4) are capable of asexual proliferation in laboratory mice. One tetrathyridial and 2 acephalic isolates from dogs proliferated asexually. Further investigation is warranted to determine which of the lineages represent distinct species and to determine the life history strategies of Mesocestoides spp.