Freshwater mussels are fundamental contributors to a healthy ecosystem, yet considered globally imperilled. During Australia’s most recent drought, mass mortality of freshwater mussels across the Murray Darling Basin (MDB) was observed in response to hot and dry conditions. As a result of climate change, the onset and intensity of such drought scenarios within this region will continue to increase, which may further deplete freshwater mussel populations. This presents an urgent need to understand freshwater mussel distribution and monitor population responses to environmental change. Species specific environmental DNA (eDNA) assays provide a sensitive and efficient method to identify and monitor distribution whilst removing several of the challenges involved in conventional detection methods. Until now, the development of such assays has been hindered by a disconnect between morphological and genetic species identification for freshwater mussels in the MDB highlighting the need for a taxonomic review. To avoid a time-consuming taxonomic review, I used barcode gap analysis and phylogenetic tree reconstruction to determine that there are three genetically distinct putative species of freshwater mussels present in the MDB. I developed robust eDNA quantitative PCR assays to detect two of these putative species. I then completed a case study on the Ovens River in Victoria to test the use of these assays in a natural environment. This allowed me to determine the probability of detection, refine systematic sampling approaches and compare the use of eDNA to common freshwater mussel detection techniques (i.e. brailing sediment using hands and feet, snorkelling where possible). These eDNA assays provide a valuable tool to monitor freshwater mussel distributions in a changing climate and establish their environmental requirements for long term targeted conservation.