The River Murray is Australia’s longest (2,500 km) river, and together with its major tributary the Darling River, supports nationally and internationally important ecosystems and socio-economic values. Our aim was to (1) analyse the water quality in this large arid river system using a long term (1978−2015) dataset collected from 24 monitoring sites from the headwaters to the Lower Lakes, and (2) assess the present and likely future state of water quality based on the data analysis and other factors such as water recovery under the Basin Plan and climate change. The River Murray’s water quality is highly variable, but on average electrical conductivity (EC), pH, turbidity, nutrient, colour and chlorophyll a levels increase with distance downstream from the headwaters to the lower reaches. This is a function of the natural accumulation of dissolved and particulate components and intermittent, mostly diffuse source, pollutant inputs. The Darling tributary inflow increased turbidity, total phosphorus and pH in the main River Murray channel. Based on long-term trend analysis at four representative sites, EC, nutrients and colour showed declining trends on average at most sites except in the headwaters. Temperature showed increases at two sites. Higher flow increased concentrations of most quality parameters, although at very high flows decreases in pH, EC, turbidity and nitrate occurred at many sites. The extreme “Millennium” drought (2002−2009) period resulted in lowered concentrations of many water quality parameters, indicating retention in the landscape. In the post-drought flooding (2010−2012) period a large amount of organic material was mobilised, resulting in much higher peak colour/carbon concentrations than when mid-range flooding was more frequent. Water recovery activities and new instructure should enable restoration of more mid-range flooding events to minimise these risks. It is considered critical that this unique monitoring program is continued as a Basin-wide water management plan is implemented.