Corydoras difluviatilis, new species, is described from the upper rio Paraná and the upper rio São Francisco, Brazil. The new taxon differs from its congeners by three exclusive features: the posterior pectoral-spine serrations restricted to its proximal half, and the parapophyses of the fourth free vertebra reduced and separated from each other, not fused into a haemal arch. The new species has several plesiomorphic character states relative to the assemblage composed of Brochis and most Corydoras species, such as absence of contact between the supraoccipital and nuchal plate, reduction of the ossified portion of hypobranchial 2, and well-developed mesial expansions of the first and second infraorbitals, indicating its basalmost position within the genus.