Analysis of environmental DNA (eDNA) is an increasingly valuable approach for detecting the presence of rare fish species. This approach allows for species detection without the need to deploy traditional sampling gear (e.g., nets, electrofishing) and is sensitive enough to detect species at very low abundance. The Australian grayling Prototroctes maraena is a nationally threatened fish whose distribution in NSW has undergone a severe southward contraction in recent decades. Australian grayling is diadromous and must migrate between freshwater and the sea to complete its life cycle. During Autumn, adult fish undertake a downstream migration from upper freshwater reaches of rivers to lowland freshwater reaches immediately above the estuary, where they congregate to spawn. We tested an eDNA sampling method to improve our ability to determine the presence of the Australian grayling within catchments. eDNA surveys were conducted in the lower reaches of three rivers in NSW (Deua, Tuross) and Victoria (Bunyip) over the spawning season (April – June). The sampling design targeted the timing and location of spawning aggregations to maximise the likelihood of detecting Australian grayling. Results from the study show that consideration of species’ ecology can effectively optimise the efficacy of eDNA for rare species detection.