Understanding how populations respond to habitat loss is central to conserving biodiversity. Population genetic approaches enable the identification of the symptoms of population disruption in advance of population collapse. However, the spatio-temporal scales at which population disruption occurs are still too poorly known to effectively conserve biodiversity in the face of human-induced landscape change. We employed microsatellite analysis to examine genetic structure and diversity over small spatial (mostly 1-50 km) and temporal scales (20-50 years) in the squirrel glider (Petaurus norfolcensis), a gliding mammal that is commonly subjected to a loss of habitat connectivity. We identified genetically differentiated local populations over distances as little as 3 km and within 30 years of landscape change. Genetically isolated local populations experienced the loss of genetic diversity, and significantly increased mean relatedness, which suggests increased inbreeding. Where tree cover remained, genetic differentiation was less evident. This pattern was repeated in two landscapes located 750 km apart. These results lend support to other recent studies that suggest the loss of habitat connectivity can produce fine-scale population genetic change in a range of taxa. This gives rise to the prediction that many other vertebrates will experience similar genetic changes. Our results suggest the future collapse of local populations of this gliding mammal is likely unless habitat connectivity is maintained or restored. Landscape management must occur on a fine-scale to avert the erosion of biodiversity.