Fish communities from four catchments in the Murray—Darling river system were analysed in relation to climate, hydrology and river regulation. Using the annual proportional flow deviation as a measure of river regulation, the Paroo River catchment was assessed as unregulated, the Darling River catchment as mildly regulated and the Murrumbidgee River and River Murray catchments as highly regulated. A total of 11010 fish, representing nine native and three alien species, was caught during high and low flow seasons in the four catchments. Native species, such as golden perch Macquaria ambigua (Percichthyidae), bony herring Nematalosa erebi (Clupeidae) and spangled perch Leiopotherapon unicolor (Teraponidae), dominated fish communities in the Paroo and Darling catchments, but alien species, mostly carp, Cyprinus carpio (Cyprinidae), were also abundant. Both native and alien species were more abundant in these catchments after flooding, but there was little change in species composition between high and low flow seasons at the catchment level. Carp dominated communities in the Murray and Murrumbidgee catchments. There was a significant trend for reduced species diversity in increasingly regulated catchments. River regulation may alter the relative abundance of native and alien fish by desynchronizing environmental cycles and the reproductive cycles of native species. Ordination of species abundances showed discrete fish communities that reflect the geographical separation between catchments. Differences between communities are related to opportunities for dispersal, the environmental tolerances of dominant species and the modifying effects of river regulation. Fish communities in lakes exhibited less seasonal variation than riverine communities within the same catchment, indicating the greater seasonal stability of lakes compared with regulated and unregulated river reaches. Management of fish resources needs to include catchment-specific strategies within current State and basin-wide management programmes.