The rarity of top predators has been identified as a contributing factor in the collapse of entire ecological systems. Elucidating the ecology, evolutionary potential and population response of species before a threatening process requires a combination of measures of phenotypic variation, demography and genetic diversity using an array of markers. Here we use such information to determine the population structure and its conservation implications for a keystone marsupial carnivore in Western Australia, the northern quoll Dasyurus hallucatus, ahead of the spread of the exotic and poisonous cane toad Chaunus marinus from the east. The western populations occur in two discrete geographic regions on the mainland, the Kimberley and Pilbara, separated by the arid Great Sandy Desert, and on several continental islands. Both mitochondrial DNA sequences and 11 nuclear microsatellite loci reveal clear differentiation of the Kimberley and Pilbara regions. Offshore islands with permanent sea-channels from the mainland show reduced diversity and were genetically distinct indicating genetic isolation, whereas those separated from the mainland by very shallow channels were more similar to the adjacent mainland populations, suggesting on-going gene flow. There was little indication that any population within the distribution of the species had experienced recent declines but there was marked variation in sexual dimorphism suggesting diversity in demographic performance. These populations in Western Australia differ from those remaining to the east in Queensland and the Northern Territory, in both genetic structure and demographic parameters and represent the last intact populations in Australia that have not experienced major declines concomitant with the spread of the cane toad. Consequently, the genetic and demographic flexibility recorded by this study provides guidance for the conservation and management of a major marsupial predator before and during its potential decline associated with an invasive, threatening prey species.