Understanding the processes that guide and determine different genetic diversity patterns in wild populations is very important for conservation, because it reveals threats and possible management measures. The pampas cat, Leopardus colocolo, is a small felid from Neotropical region, associated to open habitats. In Brazil the species occurs in unconnected areas in the midwestern and southern Brazil. Although the specie’s taxonomy is controversial, it is known that some populations are genetically structured, as a result from long periods of isolation. We analyzed 13 microsatellite loci to characterize the genetic divergence between the two geographically disconnected Brazilian populations of pampas cat. Samples from Uruguay were included, since previous studies have indicated the existence of only one population between Uruguay and southern Brazil. Our sample comprised 23 Leopardus colocolo from midwestern and 22 from southern Brazil and Uruguay. The Brazilian populations showed significant genetic structure, corresponding to two genetic clusters. The gene flow between the populations is low, and apparently not sufficient to stop genetic differentiation. Therefore, we suggest these populations should be considered as different management units. The southern Brazil and Uruguay population seems to be isolated to the north by the Atlantic Forest, and to the west and south by the Paraná and Uruguay rivers. Although its genetic diversity does not seem to be reduced, this population showed levels of inbreeding and low effective population size, which can lead to the reduction in population viability and become extremely dangerous for specie’s conservation in these areas.