AbstractEcosystems are currently changing at unprecedented rates due to anthropogenic influences. Application of appropriate management regimes and mitigation measures requires knowledge of ecological community composition and monitoring of any changes that occur. Environmental DNA‐based monitoring is becoming increasingly common and offers substantial potential as a noninvasive method associated with highly repeatable and reliable results. In this study, we monitored river systems in Western Greece that have been strongly impacted by anthropogenic activities and the spread of an alien invasive fish species, the Eastern mosquitofish (Gambusia holbrooki). This invasive species has been credited as the major cause for the drastic decline of two endemic killifish species (Valencia letourneuxi and Valencia robertae). Here, we investigated the efficacy of an environmental DNA (eDNA‐based) method of detection for all three species, as an alternative to conventional monitoring methods. Initially, a mesocosm experiment provided material for the design and validation of the sampling protocol. This was followed by two sampling periods in the field conducted in autumn 2017 and 2018, comparing the novel eDNA assays with the conventional surveying methods in six and 20 systems, respectively. eDNA detection consistently outperformed the traditional monitoring methods for both V. letourneuxi and V. robertae and was comparable for the invasive G. holbrooki. This supports the now increasing body of literature, highlighting the benefits of species‐specific, targeted eDNA assays for the assessment of threatened and/or invasive species, one which can be utilized by conservation organizations and government bodies alike. However, we note that care should always be taken when designing such tools and strict validation steps should be adhered to, particularly with respect to minimizing the probability of false positives and negatives.