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Wildlife species impacted by habitat loss and fragmentation often require conservation efforts to maintain populations. Long-nosed bandicoots (Perameles nasuta) still persist within the highly urbanised matrix of northern Sydney (Australia). One population at North Head, Sydney, is currently listed as an Endangered population due to its small size, apparent isolation and other threats. To support future management, we used 1,446 single nucleotide polymorphism markers (SNPs) from 167 bandicoots to: i) assess the assumption of isolation and determine if genetic structuring is present between North Head and individuals from 11 other localities in northern Sydney, and ii) investigate genetic diversity over time in the North Head population from 2002 to 2018. Analyses confirmed population structuring and genetic divergence between North Head and greater northern Sydney. Three distinct populations were identified that corresponded to geographic localities (North Head, northern Sydney and Mosman). All populations were significantly differentiated (FST = 0.171–0.345), suggesting local genetic drift between localities. North Head genetic diversity indices estimated between 2002 to 2018 showed relatively constant levels of allelic richness (1.90–2.00) and observed heterozygosity (HO = 0.231–0.310) along with minor levels of inbreeding (FIS 0.020–0.052). The identification of some individuals sampled on North Head that were assigned to other populations suggests some sporadic geneflow into the population has occurred and may have assisted with maintaining genetic diversity. These data suggest that the North Head population is distinct from other northern Sydney populations and has relatively constant levels of genetic diversity.