Abstract Intraspecific genetic diversity provides the evolutionary potential to adapt to changing environments and ‘hotspots’ of high intraspecific diversity are recognized as key targets for conservation. In south‐western Australia, intraspecific genetic diversity for mesic taxa is not uniformly distributed. Many species comprise highly divergent lineages with unique haplotypes resulting from contraction to refugia during historical arid cycles. Sampling strategies in studies of the region’s unique and ancient freshwater fauna have often focused on broad distributional range, making it difficult to determine boundaries between lineages and the location of genetic hotspots. This study explored the spatial distribution of intraspecific genetic diversity in the threatened freshwater mussel, Westralunio carteri. Mitochondrial DNA sequences for 164 specimens, sampled from all basins within the distribution of the species, were used to describe lineage boundaries and the location of hotspots, and to reconstruct historical demographics. There was strong evidence for three subregions of genetic diversity based on the largely non‐overlapping distributions of three evolutionarily significant units. Spatial and demographic analyses suggest that these evolutionarily significant units persisted through past arid cycles in separate refugia. The majority of haplotypes were unique to a single location, indicating limited connectivity among populations in recent times. Hotspots were identified throughout the region. Most notably, a significant hotspot in the south‐western corner probably arose through the overlap of lineages in historical refugia. Conservation assessments often focus on the species as a whole, even though sublineages, hotspots and the threats faced are not evenly distributed across the species range. This paper highlights that effective conservation of spatially structured taxa requires targeted management of multiple genetic units. Given the importance of formal taxonomic description for conservation listings, further investigation of the potential for species delimitation within W. carteri is required.