Effects of dam operation and extraction of water from rivers on spatial variation in hydrological regimes, and consequences for freshwater biodiversity, are widely predicted but seldom assessed empirically. Evidence of linkages between hydrology and beta diversity contributes to water-management decisions to support landscape-scale biodiversity and avoid inadvertently contributing to further biodiversity decline. Using six lowland rivers in Australia's Murray – Darling Basin that formed a gradient of hydrological alteration, we examined (1) spatial variation in hydrology under modelled scenarios of low water-resource development and flow modification by dams and extraction, (2) how beta diversity of fish among and within rivers was associated with spatial hydrological variation and whether patterns of overall beta diversity differed between native and non-native species, and (3) the associations of spatial and environmental variables and both recent and long-term hydrology with beta diversity. Spatial variation in hydrology among rivers was higher under the modified scenario than under the low-development scenario yet change in the magnitude of within-river (longitudinal) variation was inconsistent between rivers. Beta diversity among rivers was significantly associated with spatial variation in hydrology only in certain circumstances (native species assemblages in specific years). Within-river beta diversity varied among rivers yet was unrelated to longitudinal variation in modified hydrological regimes. Patterns of beta diversity did not differ appreciably if non-native species were included in or excluded from analyses. These findings contradict predictions adopted in ecohydrological science that water resource development homogenises hydrological regimes, in turn causing biotic homogenisation in lowland rivers.