ABSTRACTIn Estonia, three invasive North American crayfish species—Pacifastacus leniusculus, Faxonius limosus, and Procambarus virginalis—have been detected through the annual monitoring program. To protect Astacus astacus, the only native freshwater crayfish species in Estonia, rapid and effective conservation‐based management actions are necessary. Recently, the environmental DNA (eDNA) approach has been increasingly used in Europe to detect crayfish species and the crayfish plague pathogen Aphanomyces astaci. Our study explored the potential of integrating the eDNA approach into ongoing annual monitoring programs for invasive crayfish species and A. astaci. We also evaluated the relationship between eDNA concentration and signal crayfish population density at a single location. We filtered 139 eDNA samples from 16 water bodies and screened them for A. astacus, P. leniusculus, and A. astaci using singleplex qPCR assays. A subset of the samples was also screened for P. virginalis and F. limosus. Crayfish eDNA was detected in nine out of 14 water bodies where presence was confirmed by trapping, resulting in a 64% detection efficiency. Detection of P. virginalis was only observed in samples with amplifications below the limit of detection, and A. astaci eDNA was found in only one water body hosting invasive crayfish species. Although we could not establish a convincing quantitative correlation between the estimated P. leniusculus eDNA concentration and crayfish population density, we conclude that the eDNA approach is promising and, with further optimization, it can be integrated into routine monitoring of crayfish and crayfish plague pathogen as a supplement to traditional trapping methods.