We present a systematic study of Scapteromys populations from Argentina, Paraguay, and Uruguay, based on molecular and morphological evidence. First, based on DNA sequences (801 base pairs) from the mitochondrial cytochrome-b gene, we found that Scapteromys populations fall into 2 main clades; 1 formed by Argentinean and Paraguayan populations together with 1 population from western Uruguay, and the other constituted by the remaining Uruguayan populations. Analysis of molecular variance showed that 86.93% of the genetic variation uncovered in Scapteromys is explained by differences between clades. Morphological analyses corroborated the existence of 2 main morphotypes among Scapteromys specimens. The phylogeographic break identified is mostly congruent with patterns of morphological and chromosomic variation. In light of these results, we propose that S. aquaticus be elevated to the rank of species, we redefine the known distributions of S. aquaticus and S. tumidus, and we provide a list of character states that allow an unambiguously diagnosis of both species.