Knowledge of population structure and patterns of connectivity is required to implement effective conservation measures for the purple-crowned fairy-wren (Malurus coronatus), a threatened endemic of northern Australia. This study aimed to identify barriers to dispersal across the distribution of M. coronatus, investigate the impact that the recent declines may have on population connectivity, and propose conservation actions to maintain natural patterns of gene flow. Analysis of mitochondrial DNA sequences from 87 M. coronatus identified two phylogenetic clusters that corresponded with the phenotypically defined western (M. c. coronatus) and eastern (M. c. macgillivrayi) subspecies. The genetic divergence between these subspecies was consistent with isolation by a natural barrier to gene flow, and supports their separate conservation management. Within the declining M. c. coronatus, the lack of genetic divergence and only slight morphological difference between remnant populations indicates that populations were recently linked by gene flow. It is likely that widespread habitat degradation and the recent extirpation of M. c. coronatus from the Ord River will disrupt connectivity between, and dynamics within, remnant populations. To prevent further declines, conservation of M. coronatus must preserve areas of quality habitat and restore connectivity between isolated populations.