Spatial and temporal variation in fish-assemblage structure within isolated waterholes on the floodplains of Cooper Creek, Australia, was studied during the 2001 dry season, a period of natural drought in this arid-zone river. Spatial variation in fish-assemblage structure and the abundance of five species in disconnected waterholes early in the dry season (April 2001) were related to the extent of floodplain inundation 14 months previously, and to the interconnectedness of waterholes and waterhole habitat structure. As the dry season progressed, waterhole volumes decreased owing to evaporative water loss and structural habitat elements (anabranches, bars, boulders) became exposed. Marked changes in fish assemblage structure between the early (April) and late (September) dry season were related to habitat loss but not to water chemistry. Interactions between flow and habitat across a nested hierarchy of spatial scales (the floodplain, the waterhole and habitat patches within waterholes) were crucial to the persistence of fish assemblages through the 2001 dry season. We conclude that the magnitude, timing and frequency of floodplain inundation and natural variations in waterhole volume must be maintained if we wish to sustain the distinctive habitats and fish assemblages of this arid-zone floodplain river.