Variation in the nucleotide sequence of the entire mitochondrial cytochrome b gene (1140 bp) was examined for 27 individuals representing 13 species of South American rodents of the genera Ctenomys, Octodontomys, Tympanoctomys, and Spalacopus. Representatives of the family Echimyidae, Euryzygomatomys and Mesomys, were used as outgroups to test the monophyly of the Octodontinae and Ctenomyinae. Relationships among species of tuco-tucos (genus Ctenomys) were also examined including representatives of the three described subgenera and the two sperm morphs. Reciprocal monophyly of the Octodontinae and Ctenomyinae is strongly supported. Several basal relationships among species of the genus Ctenomys are poorly resolved, suggesting the possibility of a hard polytomy due to a rapid and potentially simultaneous radiation early in the history of the genus. In other cases, clades within the Ctenomyinae previously identified on the basis of allozymes, chromosomes, parasites, or skull morphology were supported. Calibrations based on the fossil record suggest that the mitochondrial cytochrome b of these caviomorphs has evolved at a rapid rate, comparable to those proposed for Mus-Rattus, and three to four times higher than ungulate rates.