Freshwater ecosystems host disproportionately high numbers of species relative to their surface area yet are poorly protected globally. We used data on the distribution of 1631 species of aquatic plant, mollusc, odonate and fish in 18,816 river and lake catchments in Europe to establish spatial conservation priorities based on the occurrence of threatened, range-restricted and endemic species using the Marxan systematic conservation planning tool. We found that priorities were highest for rivers and ancient lakes in S Europe, large rivers and lakes in E and N Europe, smaller lakes in NW Europe and karst/limestone areas in the Balkans, S France and central Europe. The a priori inclusion of well-protected catchments resulted in geographically more balanced priorities and better coverage of threatened (critically endangered, endangered and vulnerable) species. The a priori exclusion of well-protected catchments showed that priority areas that need further conservation interventions are in S and E Europe. We developed three ways to evaluate the correspondence between conservation priority and current protection by assessing whether a cathment has more (or less) priority given its protection level relative to all other catchments. Each method found that priority relative to protection was high in S and E Europe and generally low in NW Europe. The inclusion of hydrological connectivity had little influence on these patterns but decreased the coverage of threatened species, indicating a trade-off between connectivity and conservation of threatened species. Our results suggest that catchments in S and E Europe need urgent conservation attention (protected areas, restoration, management, species protection) in the face of imminent threats such as river regulation, dam construction, hydropower development and climate change. Our study presents continental-scale conservation priorities for freshwater ecosystems in ecologically meaningful planning units and will thus be important in freshwater biodiversity conservation policy and practice, and water management in Europe.