Dryland rivers are renowned for their periods of ‘boom’ related to the episodic floods that extend over vast floodplains and fuel incredible production, and periods of ‘bust’ where the extensive channel network is restricted to the permanent refugial waterholes. Many of these river systems are unregulated by dams but are under increasing pressure, especially from water abstraction and overland flow interception for agriculture and mining. Although some aquatic organisms with desiccation-resistant life stages can utilise ephemeral floodplain habitats, the larger river waterholes represent the only permanent aquatic habitat during extended periods of low or no flow. These waterholes act as aquatic refugia in an otherwise terrestrial landscape. Variable patterns of connection and disconnection in space and time are a fundamental driver of diversity and function in these dryland river systems, and are vital for dispersal and the maintenance of diverse populations, generate the spatial and temporal variability in assemblage structure for a range of different organisms and fuel the productivity that sustains higher trophic levels. Changes to natural patterns of connection and disconnection of refugial waterholes, owing to water-resource development or climate change, will threaten their persistence and diminish their functional capacity to act as aquatic refugia.