In light of the pressures on water resources in Western Australia, there is an increasing demand for independent ecological impact assessments of future supply options. This study examined the fish migration patterns of fishes in the Blackwood River and its tributaries within and outside the surface expression zone of the Yarragadee Aquifer; an identified yet controversial water supply option for Perth. Fish migration patterns, population demographics and community structure were related to a number of key environmental variables. Main channel sites receiving most groundwater discharge had much greater abundances of salt intolerant freshwater native species than those upstream of the discharge. This suggests that groundwater input in summer provides refuge habitat and this enables those populations to continue to be sustainable in the main channel. Many of the spatial migration variations were also attributed to differences in hydrological regimes of these systems. For example, Freshwater Cobbler migration strength was positively correlated with main channel base flows. The perennial Milyeannup Brook provided crucial breeding habitat for the threatened Balston’s Pygmy Perch and the species is particularly vulnerable to predicted habitat decline due to aquifer extraction. Other impacts on these fauna by changes in environmental variables are discussed.