AbstractThe hydrological variability of intermittent streams means that the spatial distribution of dry‐season aquatic refuges within river networks and the temporal dynamics of hydrological connectivity between them are critical for the persistence of aquatic biodiversity. Here, a new approach is demonstrated to identify surface water bodies as priority refuges for efficient conservation management of freshwater biodiversity in intermittent stream networks.Recently developed models of surface water extent and daily streamflow were used to represent spatio‐temporal variations in hydrological connectivity and surface water persistence within river networks of eastern Australia over a 107‐yr period. Using this information, systematic conservation planning was applied to prioritize aquatic areas for conservation of 25 fish species under two scenarios. One scenario identified priority refuges to complement those already occurring in protected areas, whereas the other did not consider protected area status.The priority networks identified concentrated on the main stems of river catchments where surface water was more likely to be persistent and aquatic refuges were more likely to be connected, but also included headwaters for rare fish species. All three set conservation targets for the 25 fish species can be met in the best solution of priority networks. Although the second scenario achieved the targets with a smaller size of priority network overall, it required more new aquatic refuges and was thus less efficient than the first scenario.The newly developed datasets are useful for freshwater conservation prioritization because they account for hydrological variability of intermittent streams. The systematic prioritization approach applied is transferable to other regions and freshwater taxa to identify aquatic refuges for biodiversity conservation within intermittent stream systems.