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Many rivers experience intermittent flows naturally or as a consequence of water abstraction. Climate change is likely to exacerbate flow variability such that dry spells may become more common. It is important to understand the ecological consequences of flow intermittency and habitat fragmentation in rivers, and to identify and protect habitat patches that provide refugia for aquatic biota. This paper explores environmental factors influencing dry season fish losses from isolated waterbodies in Cooper Creek, an unregulated arid-zone river in the Lake Eyre Basin, Australia. Multivariate ordination techniques and classification and regression trees (CART) were used to decompose species-environment relationships into a hierarchically structured data set, and to determine factors explaining changes in fish assemblage structure and species losses over a single dry season. Canonical correspondence analysis (CCA) explained 74% of fish assemblage change in terms of waterhole morphology (wetted perimeter, depth), habitat structure (bench development, off-take channels), waterhole quality (eroded banks, gross primary production), the size of surrounding floodplains and the relative isolation of waterholes. Classification trees for endemic and restricted species reaffirmed the importance of these waterhole and floodplain variables as drivers of fish losses. The CCA and CART models offer valuable tools for identification of refugia in Cooper Creek and, possibly, other dryland rivers.