Aim: Refugia play a key ecological role for the persistence of biodiversity in areas subject to natural or human disturbance. Temporary freshwater ecosystems regularly experience dry periods, which constrain the availability of suitable habitats. Current and future threats (e.g. water extraction and climate change) can exacerbate the negative effects of drying conditions. This could compromise the persistence of a large proportion of global freshwater biodiversity, so the identification and protection of refugia seems an urgent task. Location: Northern Australia. Methods: We demonstrate a new approach to identify and prioritize the selection of refugia and apply it to the conservation of freshwater fish biodiversity. We identified refugia using estimates of water residency time derived from satellite imagery and used a systematic approach to prioritize areas that provide all the fish species inhabiting the catchment with access to a minimum number of refugia while maximizing the length of stream potentially accessible for recolonization after the dry period. These priority refugia were locked into a broader systematic conservation plan with area-based targets and direct connectivity. We accounted for current threats during the prioritization process to ensure degraded areas were avoided, thus maximizing the ecological role of priority refugia. Results: Priority refugia were located in lowland reaches, where the incidence of threats was less prominent in our study area and headwaters in good condition. An additional set of 106 planning units (6500 km 2 ) were required to represent 10% of each species' distribution in the broad conservation plan. A hierarchical management zoning scheme was applied to demonstrate how these key ecological features could be effectively protected from the major threats caused by aquatic invasive species and grazing. Main conclusions: This new approach to identifying priority refugia and incorporating them into the conservation planning process in a systematic way would help enhance the resilience of freshwater biodiversity in temporary systems.