AbstractThe white‐clawed crayfish (Austropotamobius pallipes) is an emblematic taxon of European rivers, found mainly in oxygenated streams, known to be an excellent indicator of river quality. Since several decades, the population of A. pallipes declined in relation to anthropogenic pressure, habitat loss, and competition with pests (invasive crayfish, crayfish plague). This endangered species is now submitted to conservation strategies by freshwater managers in order to survey and protect the remaining populations. In France, traditional surveys in freshwater environments were performed by electric fishing, kick‐net fishing, or trapping, particularly disruptive for the environment and very time‐consuming. However, with the rise of molecular genetic technology, new methods based on the detection of environmental DNA (eDNA) have emerged. We present here the results of an optimized study for the detection of the endangered crayfish Austropotamobius pallipes in France, considering certain environmental co‐factors and comparing two PCR methods (qPCR and ddPCR). After improving laboratory procedures, we were able to detect the presence of the crayfish up to 2 km downstream from a known point of presence and unfortunately highlight the disappearance of a historical population, after sampling two consecutive years. Such a level of precision is interesting because it makes it possible to precise the presence of specimens in a relatively restricted area and to orient traditional prospecting, necessary for certain additional studies. During our study, we observed better probabilities of detection during the summer period, but in a growing context of climate change, we advise to adapt the sampling year by year. That said, this methodology is a very useful tool for the detection of rare and/or endemic species and we did not observe any difference between the two PCR methods used.