Hydrological regime, physical habitat structure and water chemistry are interacting drivers of fish assemblage structure in floodplain rivers throughout the world. In rivers with altered flow regimes, understanding fish assemblage responses to flow and physico-chemical conditions is important in setting priorities for environmental flow allocations and other river management strategies. To this end we examined fish assemblage patterns across a simple gradient of flow regulation in the upper Murray–Darling Basin, Australia. We found clear separation of three fish assemblage groups that were spatially differentiated in November 2002, at the end of the winter dry season. Fish assemblage patterns were concordant with differences in water chemistry, but not with the geomorphological attributes of channel and floodplain waterholes. After the summer-flow period, when all in-channel river sites received flow, some floodplain sites were lost to drying and one increased in volume, fish assemblages were less clearly differentiated. The fish assemblages of river sites did not increase in richness or abundance in response to channel flow and the associated potential for increased fish recruitment and movement associated with flow connectivity. Instead, the more regulated river's fish assemblages appeared to be under stress, most likely from historical flow regulation. These findings have clear implications for the management of hydrological regimes and the provision of environmental flows in regulated rivers of the upper Murray–Darling Basin. Copyright © 2010 John Wiley & Sons, Ltd.