Context: A core theme in ecohydrology is understanding how hydrology affects spatial variation in the composition of species assemblages (i.e., beta diversity). However, most empirical evidence is from research in upland rivers spanning small spatial extents. Relatively little is known of the consequences of hydrological variation for beta diversity across multiple spatial scales in lowland rivers. Objectives: We sought to examine how spatial variation in hydrology and fish beta diversity within and among rivers changed over time in response to intensification and cessation of hydrological drought. Methods: We used monitoring data of fish assemblages, coupled with hydrological and biophysical data, to test how spatial variation in hydrology and multiple components of fish beta diversity in lowland rivers of the Murray—Darling Basin (Australia) varied across spatial scales during contrasting hydrological phases. Results: Spatial variation in hydrology among rivers declined with increasing duration of drought before increasing during a return to above-average flows. Spatial variation in hydrology within rivers did not show consistent changes between hydrological phases. Beta diversity among and within rivers showed variable, river-specific changes among hydrological phases for both incidence- and abundance-based components of assemblage composition. Conclusions: Inconsistent hydrology—beta diversity patterns found here suggest that mechanisms and outcomes of drought and flooding impacts to beta diversity are context-dependent and not broadly generalisable. Our findings indicate that hydrological fluctuations occurring in the Murray—Darling Basin in the period analysed here did not cause significant or consistent homogenisation or differentiation of freshwater fish assemblages.