The Malleefowl ( Leipoa ocellata ) is a threatened megapode bird that persists on the Eyre Peninsula of South Australia—an area that has undergone substantial clearance of native vegetation over the past 200 years. Habitat loss and fragmentation can negatively affect long-term conservation status by creating small and isolated subpopulations that lead to reduced gene flow, increased genetic drift, and inbreeding. In this study, we test whether Malleefowl on the Eyre Peninsula exhibit population structure that may have resulted from recent anthropogenic land cover changes. We used reduced-representation high-throughput DNA sequencing to obtain a dataset of 17,851 single nucleotide polymorphisms (SNPs). Our results suggest that Malleefowl are not genetically homogeneous across the Eyre Peninsula and that habitat fragmentation has likely driven differentiation of at least two distinct populations by decreasing opportunities for gene flow. We also provide preliminary genetic evidence for female-biased dispersal in Malleefowl. Our study highlights the potential problems caused by anthropogenic land cover changes for threatened Malleefowl populations and underscores the need for ongoing monitoring. We emphasise the importance of both large continuous native vegetation patches and small connecting habitat fragments even in highly fragmented landscapes. We encourage land managers to facilitate between-patch movement by improving native vegetation cover and controlling overabundant herbivores to improve habitat quality by allowing natural regrowth of existing native vegetation. • Habitat fragmentation has driven differentiation of Malleefowl populations on the Eyre Peninsula. • Preliminary genetic evidence for female-biased dispersal in a megapode. • Malleefowl management decisions should involve genetic targets. • Managers should facilitate between-patch gene flow by improving vegetation cover. • Lacking improvement, translocations may be necessary for better genetic health.