Arboreal gliders are vulnerable to habitat fragmentation and to barriers that extend their glide distance threshold. Habitat fragmentation through deforestation can cause population isolation and genetic drift in gliding mammals, which in turn can result in a loss of genetic diversity and population long-term persistence. This study utilised next generation sequencing technology to call 8784 genome-wide SNPs from 90 sugar gliders (Petaurus breviceps) sensu stricto. Samples were collected from 12 locations in the Lake Macquarie Local Government Area (New South Wales). The sugar gliders appeared to have high levels of gene flow and little genetic differentiation; however spatial least cost path analyses identified the Pacific Motorway as a potential barrier to their dispersal. This Motorway is still relatively new (<40 years>old), so man-made crossing structures should be erected as a management priority to mitigate any long-term effects of population isolation by assisting in the dispersal and gene flow of the species.