The flow regime of the River Murray has changed markedly over the last century, and especially the last 50 years, through increased diversions, construction of dams, weirs and levees and changes in operational procedures. A model developed by the Murray–Darling Basin Commission is used to compare simulated natural (unregulated) flows at eight stations with those at seven consecutive stages in the development of regulation. Monthly and annual average flows and coefficients of variation and skewness were computed, and the flow‐duration, peak‐flow and low‐flow characteristics curves plotted. The results confirm that average monthly and annual flows are now considerably lower than those which prevailed under natural conditions. The seasonal distribution of flows has changed in the upper Murray, owing to the influence of dams. Flow‐duration characteristics now vary considerably along the river, whereas there was little change under natural conditions. The effect of regulation on flow‐duration characteristics is minimal at Albury and becomes more pronounced downstream; it is most apparent in regard to flows exceeded 20–80% of the time. The magnitude of average annual floods (annual exceedance probability 50%) has been reduced by over 50% at all stations, but big floods (average recurrence interval 20 years or more) are little affected. Further, the low flows for a given annual non‐exceedance probability are higher under regulated conditions than those under natural conditions. These changes have profound implications for communities of native plants and animals in both riverine and floodplain environments, and also for the long‐term utility of the river as a resource.