Carnivores are decreasing globally due in part to anthropogenic ecological disturbances. In Argentina, human activities have fragmented wildlife habitat, thereby intensifying puma–livestock conflict and leading to population control of the predator species by hunting. We investigated genetic variability and population structure of pumas (Puma concolor) from three south-central Argentine provinces with two different management policies for the species: full protection versus legal hunting. All genetic estimates were based on 83 individuals genotyped at 25 species-specific microsatellite loci. The overall genetic diversity was high (observed heterozygosity = 0.63), but lower than in other South American populations. Spatial analyses revealed the presence of two bottlenecked genetic clusters with very similar diversity and low gene flow (3% per generation) between them. However, analyses based on a priori separated groups showed that gene flow follows increasing values of hunting pressure, converging to the area with the greatest number of individuals harvested. Our results suggest that hunting pressure likely is contributing to the gene flow pattern, limiting pumas' movements and creating a metapopulation dynamic among geographic subpopulations. Integrated demographic and genetic approaches are needed to better understand pumas' movements across the landscape and adopt successful management plans to achieve long-term population viability.