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The stone crayfish, <i>Austropotamobius torrentium</i>, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of <i>A. torrentium</i> and <i>A. pallipes</i> environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by <i>A. torrentium</i>, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the <i>A. pallipes</i> probe. The presence of <i>A. torrentium</i> is confirmed at 6 out of 15 sites. At four of these sites, <i>A. torrentium</i> is detected for the first time. In contrast, the presence of <i>A. torrentium</i> was not detected at two sites already known to harbour the species. Finally, the presence of <i>A. pallipes</i> was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (<i>A. pallipes</i>) through eDNA analysis.