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The endorheic Lake Eyre Basin drains 1.2 million square kilometres of arid central Australia, yet provides habitat for only 30 species of freshwater fish due to the scarcity of water and extreme climate. The majority are hardy riverine species that are adapted to the unpredictable flow regimes, and capable of massive population booms following heavy rainfall and the restoration of connectivity between isolated waterholes. The remainder are endemic specialists from isolated springs with very restricted ranges, and many are listed under relevant state and national endangered species legislation and also by the International Union for Conservation of Nature (IUCN). For these spring communities, which are sustained by water from the Great Artesian Basin, survival is contingent on suitable habitat persisting alongside extractive mining, agriculture and the imposition of alien species. For the riverine species, which frequently undertake long migrations into ephemeral systems, preservation of the natural flow regime is paramount, as this reinstates riverine connectivity. In this study, fish were sampled from the Bulloo River in the east to the Mulligan River in the west, along a temporal timeframe and using a standard set of sampling gears. Fish presence was influenced by factors such as natural catchment divides, sampling time, ephemerality and the occurrence of connection flows and flooding. Despite the comparatively low diversity of species, the aquatic systems of this isolated region remain in good ecological condition, and as such they offer excellent opportunities to investigate the ecology of arid water systems. However, the presence of both endangered species (in the springs) and invasive and translocated species more widely indicates that active protection and management of this unique area is essential to maintain biodiversity and ecosystem integrity.