Studies of genetic differentiation in fragmented environments help us to identify those landscape features that most affect gene flow and dispersal patterns. Particularly, the assessment of the relative significance of intrinsic biological and environmental factors affecting the genetic structure of populations becomes crucial. In this work, we assess the current dispersal patterns and population structure of Ctenomys “chasiquensis”, a vulnerable and endemic subterranean rodent distributed on a small area in Central Argentina, using 9 polymorphic microsatellite loci. We use landscape genetics approaches to assess the relationship between genetic connectivity among populations and environmental attributes. Our analyses show that populations of C. “chasiquensis” are moderately to highly structured at a regional level. This pattern is most likely the outcome of substantial gene flow on the more homogeneous sand dune habitat of the Northwest of its distributional range, in conjunction with an important degree of isolation of eastern and southwestern populations, where the optimal habitat is surrounded by a highly fragmented landscape. Landscape genetics analysis suggests that habitat quality and longitude were the environmental factors most strongly associated with genetic differentiation/uniqueness of populations. In conclusion, our results indicate an important genetic structure in this species, even at a small spatial scale, suggesting that contemporary habitat fragmentation increases population differentiation.