Fish in dryland rivers must cope with extreme variability in hydrology, temperature and other environmental factors that ultimately have a major influence on their patterns of distribution and abundance at the landscape scale. Given that fish persist in these systems under conditions of high environmental variability, dryland rivers represent ideal systems to investigate the processes contributing to and sustaining fish biodiversity and recruitment in variable environments. Hence, spatial and temporal variation in fish assemblage structure was examined in 15 waterholes of the Warrego River between October 2001 and May 2003. Fish assemblages in isolated waterholes were differentiated at the end of the dry 2001 winter but were relatively similar following high summer flows in January 2002 as a consequence of high hydrological connectivity among waterholes. Small, shallow waterholes supported more species and higher abundances than large-deep waterholes. Large, deep waterholes provided important refuge for large-bodied fish species such as adult yellowbelly, Macquaria ambigua, and the eel-tailed catfish, Tandanus tandanus. Recruitment patterns of bony bream (Nematalosa erebi), Hyrtl’s tandan (Neosilurus hyrtlii) and yellowbelly were associated with high flow events and backwater inundation; however recruitment of yellowbelly and bony bream was also evident following a zero-flow period. Departures from typical flood-induced seasonal spawning patterns may reflect opportunistic spawning behaviours appropriate to the erratic patterns of flooding and dry spells in dryland rivers.