Understanding the spatial structure of populations is important in developing effective management options for threatened species, and for managing habitat connectivity for metapopulation function, and for demographic and genetic heterogeneity. We used genetic information to investigate the structure of populations of the quokka, Setonix brachyurus, in south-west Western Australia. We hypothesised that movement between known populations would be relatively rare and result in significant genetic structuring. Genetic analyses from 412 adult individuals at 14 nuclear markers (microsatellite) from 33 sampling locations identified structure, diversity and spatial separation of quokkas across their mainland distribution and on two islands. We identified nine inferred (K = 9) populations of quokka that would be otherwise difficult to define with standard ecological techniques. The highest genetic diversity was evident in a large central population of quokka in the southern forest area and genetic diversity was lower at the peripheries of the distribution. The Rottnest Island population contained 70% of the genetic diversity of the mainland populations but the genetic diversity of animals on Bald Island was markedly lower. Populations of quokka in the northern jarrah forest were the only ones to show evidence of recent or long-term population bottlenecking. Of particular interest was the recently identified population at the Muddy Lakes area (the only remaining locality on the Swan Coastal Plain), which was identified as being genetically associated with the southern forest population. Overall, spatial and population cluster analysis showed small insular populations in the northern jarrah forest area, but in the southern forests there appears to be a large panmictic population.