Environmental DNA (eDNA) has been recently implemented as a powerful method for the detection and monitoring of freshwater species, particularly for endangered species, where eDNA methods have shown to be less invasive than other methods (e.g. electrofishing).

In this study, we developed and validated, under controlled conditions, a new species-specific tool for detecting A. anguilla from water samples by means of eDNA. Furthermore, we applied a semi-quantitative approach for monitoring of glass eel at different depths (surface and bottom) during different seasons in two rivers of northern Spain (Nalón and Sella rivers). We detected a significantly higher proportion of positive DNA amplifications in bottom than surface samples. Moreover, the proportion of replicas with positive amplification varied along the estuaries sections examined, and especially in the different sampling months. The temporal detection trends found in this study were compatible with the known upstream migration pattern of this species.