The swamp rats are distributed in Argentina, southern Paraguay, Uruguay and southern Brazil, with two species currently accepted: Scapteromys aquaticus Thomas, 1920 and Scapteromys tumidus Waterhouse, 1837. While S. aquaticus occurs in Argentina, Paraguay and western Uruguay, S. tumidus occurs in Brazil and Uruguay. Here we report for the first time the occurrence of S. aquaticus in gallery forest remnants in Southern Brazil. Karyologic analysis showed 2n = 32 and FNa = 40. Phylogenetic analyses, based on DNA sequences from the mitochondrial cytochrome-b gene indicate that the Brazilian and the Argentinian specimens of S. aquaticus shared one haplotype, while median joining analysis showed lack of population structure. This register, plus the karyotype data available for Brazilian population, recovered four karyomorphotypes in Brazil, corresponding to the two known species of Scapteromys and two unnamed species. This scenario indicates that more multidisciplinary studies are necessary to understand the actual diversity of Scapteromys.