Habitat loss and fragmentation are key threats to local koala (Phascolarctos cinereus) populations. Broad-scale management is suboptimal for koalas because distribution models are not easily generalised across regions. Therefore, it is imperative that data relevant to local management bodies are available. Genetic data provides important information on gene flow and potential habitat barriers, including anthropogenic disturbances. Little genetic data are available for nationally significant koala populations in north-eastern New South Wales, despite reported declines due to urbanisation and habitat loss. In this study, we develop 14 novel microsatellite loci to investigate koala populations in north-eastern New South Wales (Port Macquarie, Coffs Harbour, Tyagarah, Ballina) and south-eastern Queensland (Coomera). All locations were significantly differentiated (FST≤0.096-0.213; FST≤0.282-0.582), and this pattern was not consistent with isolation by distance (R2≤0.228, P≤0.058). Population assignment clustered the more northern populations (Ballina, Tyagarah and Coomera), suggesting contemporary gene flow among these sites. For all locations, low molecular variation among (16%) rather than within (84%) sites suggests historical connectivity. These results suggest that koala populations in north-eastern New South Wales and south-eastern Queensland are experiencing contemporary impediments to gene flow, and highlight the importance of maintaining habitat connectivity across this region.