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Phylogenetic analyses based on the complete nucleotide sequence of the mitochondrial 12S ribosomal RNA gene were performed for representatives of major North American cyprinid clades. These analyses resolved four monophyletic groups: (1) Western Clade, represented by Acrocheilus and Gila, (2) Creek Chub Clade, represented by Couesius, Hemitremia, Semotilus, and Margariscus, (3) Open Posterior Myodome Clade, represented by Campostoma, Phenacobius, Cyprinella, Notropis, Platygobio, and Rhinichthys, and (4) Plagopterin Clade, represented by Snyderichthys, Lepidomeda, and Meda. The overall results of these analyses refute previous hypotheses of relationships of North American Cyprinidae based on morphological data. Our analyses support recognition of Snyderichthys, formerly considered a subgenus of Gila, and indicate that the Plagopterin Clade is sister to the Creek Chub Clade. Furthermore, this analysis indicates that hypotheses of relationship between plagopterins and other Western taxa are unsupported and the Open Posterior Myodome Clade is a monophyletic group.