The collared tuco-tuco, Ctenomys torquatus Lichtenstein, 1830 (Ctenomyidae), is a subterranean rodent that occurs in grassland habitats of southern Brazil and northern Uruguay. A population of collared tuco-tucos located in Alegrete Municipality, Rio Grande do Sul State, Brazil, has been proposed as a potential new species because of their remarkable differences in chromosome number and their unique patterns of pelage coloration. The aim of this work was to evaluate the degree of genetic differentiation of this population using mitochondrial DNA (mtDNA) sequences and nuclear microsatellite markers, and to describe spatial patterns of genetic diversity for the other 5 populations of C. torquatus in southern Brazil, focusing on patterns of intra- and interpopulation variation in coat color. The analysis of 1,110 base pairs of the mtDNA cytochrome-b (Cytb) gene and 9 nuclear microsatellite loci revealed 7 haplotypes (n  =  65) and 48 alleles (n  =  70), respectively. Genetic diversity was moderately low within populations (H_E  =  0.40–0.56), and significantly partitioned among locations (R_ST  =  0.21; P < 0.01). Analysis of the microsatellite data suggested that genetic differentiation is consistent with a simple model of isolation by distance (r  =  0.56, P < 0.05), and that the population is in equilibrium between gene flow and local genetic drift. The partially reconstructed phylogeny revealed that the haplotypes derived from the Alegrete population were not reciprocally monophyletic, and that there was a lack of structure for coat color and karyotype variation. Thus, the individuals from the Alegrete population fall within the range of variation for C. torquatus, and should not be considered a new species. We suggest that they be considered a local, specialized lineage that could be treated and managed from a conservation perspective as a Management Unit.