Riverine barriers have been associated to genetic diversification and speciation of several taxa. The Rio São Francisco is one of the largest rivers in South America, representing the third largest river basin in Brazil and operating as a geographic barrier to gene flow of different taxa. To evaluate the influence of the Rio São Francisco in the speciation of small rodents, we investigated the genetic structure of Calomys expulsus with phylogenetic and network analyses of cytochrome b DNA. Our results suggested that C. expulsus can be divided into 3 subpopulations, 2 on the left and another one on the right bank of this river. The time of divergence of these subpopulations, using a Bayesian framework, suggested colonization from the south to the north/northeast. Spatial analysis using a clustering method and the Monmonier's algorithm suggested that the Rio São Francisco is a biogeographic barrier to gene flow and indicated that this river may play a role in the incipient speciation process of these subpopulations.