Variation in river flow is a strong determinant of movement behaviour for many freshwater fish species, enabling them to complete key aspects of their life cycle. As flow regimes have been altered to meet human water demands, environmental flows are used in many regulated rivers as a tool to restore aspects of the natural flow regime to benefit riverine species. A major challenge for environmental flow science and management is the availability of robust and generalisable knowledge quantifying ecological responses to flow variations. To investigate the transferability of hydro-ecological relationships between rivers, we evaluated the effects of variations in hydrology on fish movements and compared the behaviours of fish in river systems with contrasting degrees of river regulation and fragmentation. We used broad-scale acoustic telemetry to track the long-term movements of non-migratory freshwater catfish (Tandanus tandanus; n = 87) and two congeneric species of freshwater cod (Maccullochella ikei and M. peelii; n = 31 and 40, respectively) in the unregulated Nymboida River and the highly regulated Gwydir River system. We found shorter and less frequent movements, and lack of pronounced relationship with flow, exhibited by fish in the unregulated Nymboida River compared to the more mobile fish in the regulated Gwydir River system. This suggests that characteristics of flow regime variability and alteration are key determinants of intra- and interspecific variation in movement behaviours of fish. Fish in the regulated rivers showed increased movement during elevated river discharge and environmental flow release periods. In such rivers, environmental flows may benefit non-migratory species by facilitating rather than cueing breeding or other movements, allowing individuals improved connectivity to, and inundation of, higher quality habitats. Our study exemplifies the challenge of defining generalisable environmental flow rules to inform river management and highlights the importance of evaluating species- and river-specific hydro-ecological relationships.