The Neotropics harbors a high diversity of species and several hypotheses have been proposed to account for this pattern. However, while species of forested domains are frequently studied, less is known of species from open vegetation formations occupying, altogether, a larger area than the Amazon Forest. Here we evaluate the role of historical barriers and the riverine hypothesis in the speciation patterns of small mammals by analyzing an ancient rodent lineage (Thrichomys, Hystricomorpha). Phylogenetic and biogeographic analyses were carried out with mitochondrial and nuclear DNA markers to analyze the evolutionary relationships between Thrichomys lineages occurring in dry domains along both banks of the Rio São Francisco. This river is one of the longest of South America whose course and water flow have been modified by inland tectonic activities and climate changes. Molecular data showed a higher number of lineages than previously described. The T. inermis species complex with 2n = 26, FN = 48 was observed in both banks of the river showing a paraphyletic arrangement, suggesting that river crossing had occurred, from east to west. A similar pattern was also observed for the T. apereoides complex. Thrichomys speciation occurred in Late Miocene when the river followed a different course. The current geographic distribution of Thrichomys species and their phylogenetic relationships suggested the existence of frequent past connections between both banks in the middle section of the Rio São Francisco. The extensive palaeodune region found in this area has been identified as a centre of endemism of several vertebrate species and is likely to be a center of Thrichomys diversification.