The brush-tailed phascogale (Phascogale tapoatafa) is considered locally rare and vulnerable, despite being found in all mainland states of Australia. It is rarely detected in faunal surveys and the two most immediate conservation requirements are a determination of its current range and clarification of its taxonomic status. Measures of genetic differentiation amongst Phascogale tapoatafa populations in eastern, western and northern Australia were estimated using a partial (348 bp) sequence of mitochondrial DNA (cytochrome b gene). Observed sequence divergence within P. tapoatafa was substantial, with an average of 13% separating the allopatric populations in south-eastern, south-western and northern Australia. In comparison, an average of 16% sequence divergence separated the two currently recognised Phascogale species (P. tapoatafa and P. calura). Thus, Phascogale comprises four highly divergent lineages, suggesting that the genus is more diverse than previously thought. These data indicate that further taxonomic research is warranted.