Abstract The white-clawed crayfish Austropotamobius pallipes has undergone extensive declines within its native range in the last century. Because of its threatened status, European legislation requires the species to be regularly monitored and that Special Areas of Conservation (SACs) be designated for it. Knowledge on the distribution of this species is vital for addressing these needs. This study presents an environmental (e)DNA assay to detect A. pallipes in water samples, based on the mitochondrial cytochrome oxidase I (COI) gene, utilizing species-specific primers, a minor groove binding (MGB) probe and quantitative PCR. The results of this study indicate that eDNA is an effective tool for detecting A. pallipes in a lotic system, and could provide a valuable, non-invasive method for determining the distribution of this species.