One of the most severe anthropogenic impacts on river systems worldwide has been alterations to the natural flow regime. Understanding biological responses to altered flow regimes is critical to effectively rehabilitate aquatic ecosystems. This study investigated changes in the larval fish assemblages during varying hydrological conditions over 5years in the lower River Murray, in south-eastern Australia. Larval fish were sampled during spring/summer during three distinct hydrological periods: under a within-channel flow pulse and a water level raising (2005); during a drought with very low flows and stable water levels (2006, 2007 and 2008); and during an overbank flow (2010). Data were analysed for annual, spatial and seasonal variations, and correlations were examined between changes in larval assemblages and environmental variables. Hydrology was a key driver in inter-annual variation in larval fish assemblages. High abundances of small-bodied to medium-bodied native species were recorded under low flows, while for other species, abundance was more strongly correlated to temperature, representing seasonal variation. Two large-bodied species were only recorded during the within-channel flow pulse and overbank flow conditions, and significant increases in larval abundances were recorded during overbank flow conditions. We suggest groupings based on species response to hydrology (low-flow to medium-flow spawners, high-flow spawners or seasonal spawners). This study suggests that a range of conditions (low flows and overbank flows) are required to maintain a diverse and abundant native fish fauna in the lower River Murray.