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The episodic, seasonal hydrology of Lake Eyre Basin (LEB) rivers produce a series of waterholes which provide critical aquatic refugia and essential water supply during predominantly dry periods. This study used direct measures of water loss, regional meteorological data, and waterhole bathymetry to develop calibrated waterhole persistence models in a range of waterhole types throughout the Queensland portion of the LEB. Evaporation was a major driver of waterhole persistence, whereas the influence of other water balance parameters (e.g. groundwater discharge, seepage, vegetation utilisation) varied between waterholes and years. Estimates of waterhole persistence were extrapolated to the regional scale using a modelled time-series of waterhole level data and Landsat satellite imagery (1988–2019). Strong relationships were calculated between waterhole level and waterhole area (r2 = 0.88) and waterhole level and number of waterholes (r2 = 0.79) for all regional waterhole zones. Using these relationships and predicted evaporation rates under three possible future greenhouse gas scenarios, modelling showed that, by 2070, the number of persistent waterholes could reduce by up to 67% over 12 month period without flow. Similarly, reductions in waterhole area of up to 72% are possible under drier climate scenarios. These results potentially represent a significant risk to the aquatic ecosystems and other waterhole-dependent users for an already limited resource. • Evaporation is a major driver of Lake Eyre Basin waterhole persistence. • Waterhole persistence is modelled at an individual waterhole and regional scale. • Predicted higher evaporation rates under future climate change scenarios highlights significant reductions in waterhole persistence. • Estimated reductions represent a risk to aquatic ecosystems and other waterhole-dependent users. • Results highlight the need for climate change adaptation and management strategies that support waterhole sustainability. [ABSTRACT FROM AUTHOR]