Understanding the links between river flows and the health of native freshwater fish populations is critical for efficient and effective delivery of environmental water. Currently the status of freshwater fish populations is assessed primarily by examining changes in abundance and population age structure. While this data can provide some information on short-term population responses to environmental flows, critical processes such as population connectivity and genetic viability can be poorly represented. Genomic data, which require less-intensive field sampling to obtain than population demographic data, can improve information about these critical processes. Our study uses highly resolving genomic methods to explore the effects of river flow conditions on connectivity and genetic health of two Australian freshwater fish species with contrasting life histories: golden perch and Australian smelt. We demonstrate the value of genomic tools for understanding the spatial extent of population connectivity across the southern Murray-Darling Basin and the role of environment, including river flows, in shaping processes critical for long-term persistence of native fish populations.