The abundance and survival of fish in the ephemeral rivers of northern Australia are highly dependent on the existence of suitable in-stream habitat. This is particularly so during the dry season, when these rivers often break up into a series of waterholes whose water quality can affect habitat suitability for fish growth, reproduction and ultimately population size. Waterhole water quality can, in turn, be affected by hydrological connectivity, both during the wet season and as the dry season progresses. This paper describes recent data on the water quality (e.g. nutrient content, turbidity, dissolved oxygen concentration and temperature) and connectivity of waterholes in northern Australian catchments (Flinders and Gilbert River catchments) and discusses how these may be affecting observed fish species. Examples are given of how key water quality parameters (e.g. waterhole temperature) may affect the suitability of waterholes for fish growth and survival. The data and modelling techniques described provide important information for the management of northern Australian rivers as they come under increasing pressure from climate change and development (e.g. agricultural, dam construction, mining).