Australia’s natural temperate grasslands have diminished to 0.5 % of their former area since European settlement and, as a consequence, are highly fragmented and modified. Many vertebrate species that live in temperate grasslands are habitat specialists and therefore are at risk of decline through habitat loss and fragmentation. The grassland earless dragon (Tympanocryptis pinguicolla) is one such species. Once widespread, T. pinguicolla is now restricted to two general locations; the first is near Canberra in the Australian Capital Territory (including some adjacent land near Queanbeyan), and the second is the Monaro Tablelands in New South Wales. Here, we use microsatellite DNA data collected from the largest remaining populations near Canberra to examine genetic structure in this species in the context of the rapidly expanding urban landscape in this region. Our study revealed that, despite separation by only relatively small distances (largest distance*13 km), the T. pinguicolla populations are highly genetically structured with little admixture. Our analyses also revealed that the population with the largest census size, but which has recently crashed in population size, exhibited little detectable gene flow to other populations and is essentially isolated. Our data indicate that significant barriers to dispersal exist among the remaining T. pinguicolla populations and that management of this species cannot rely on natural dispersal to bolster declining populations. Many different agencies and landholders are responsible for the protection of these remnant populations and a co-ordinated effort is required to provide reasonable confidence that the species will persist.