Abstract Red-nosed mice (genus Wiedomys) are restricted to the Brazilian Cerrado and Caatinga biomes, distributed along the São Francisco River (SFR). Until recently, Wiedomys was considered monotypic, but 2 species with poorly defined geographic and morphological limits have been recognized: W. cerradensis and W. pyrrhorhinos. Our study used morphological, phylogenetic, and cytogenetic analyses to review and detail species limits within the genus and test whether the SFR could be associated with divergence between lineages. This integrative approach indicated that the SFR delineates the geographic boundaries between species, with W. pyrrhorhinos on the right bank and W. cerradensis on the left bank. The species are reciprocally monophyletic and diverge in cranial size, nasal morphology, and facial pelage color pattern. Karyotypic differences between and within W. pyrrhorhinos and W. cerradensis were also detected, and new chromosome complements (2n = 62, FN = 96 and 2n = 62, FN = 88, respectively) were found. Divergence time (0.339 to 0.025 Ma) is concordant with the timing of diversification of other co-distributed vertebrate taxa, and close to a younger estimate for the formation of SFR’s recent lower course to the east coast. Therefore, our study considerably expands knowledge of the geographic range of the genus, diagnoses of species, karyotypic variability between and within species, and reinforces the role of SFR as an important vicariant barrier in northeastern Brazil.