Flow regimes play a critical role in determining the distribution and abundance of aquatic biota, and the ecological integrity of floodplain rivers. From 2015 to 2024, we investigated temporal variation in fish assemblage structure and population demographics of key species in the main channel of the highly regulated Lower Murray River, South Australia. Across the sampling period, eight large-bodied and seven small-bodied species were sampled by boat electrofishing and fyke nets, respectively, with Nematalosa erebi (59–97% catch/annum) and Hypseleotris spp. (42–94%) being the most abundant. Assemblage structure was similar during years of low in-channel flows (<20,000 ML/d) but varied greatly during years of high in-channel (20,000–35,000 ML/d, i.e. 2022 and 2024) and overbank (>45,000 ML/d, i.e. 2017 and 2023) flows. These differences were primarily driven, during periods of elevated flow, by high abundance of exotic Cyprinus carpio and Carassius auratus and low abundance of native small-bodied species (e.g. Hypseleotris spp.). Additionally, episodic recruitment of native Macquaria ambigua was coincident with high in-channel and overbank spring–summer flows. Alternatively, recruitment of Maccullochella peelii, and to a lesser extent Bidyanus bidyanus, occurred on an almost-annual basis. Prolonged flows <20,000 ML/d in the Lower Murray River promote hydraulic and structural habitat (e.g. submerged vegetation) that favour small-bodied generalist species, while flows >20,000 ML/d facilitate a shift towards more natural riverine hydraulics that favour flow-dependent species. Increased variability in the annual flow regime that includes high in-channel or overbank flows will lead to increased recruitment of a range of species with different life histories, promoting diverse fish assemblages and resilience in populations. This provides a context for restoration of aspects of the natural flow regime in the Lower Murray River and other regulated lowland rivers.