Radon (222Rn) and major ion geochemistry were used to define and quantify the catchment-scale groundwater-surface water interactions along the Ovens River in the southeast Murray-Darling Basin, Victoria, Australia, between September 2009 and October 2011. The Ovens River is characterized by the transition from a single channel within a mountain valley in the upper catchment to a multi-channel meandering river on flat alluvial plains in the lower catchment. Overall, the Ovens River is dominated by gaining reaches, receiving groundwater from both alluvial and basement aquifers. The distribution of gaining and losing reaches is governed by catchment morphology and lithology. In the upper catchment, rapid groundwater recharge through the permeable aquifers increases the water table. The rising water table, referred to as hydraulic loading, increases the hydraulic head gradient toward the river and hence causes high baseflow to the river during wet (high flow) periods. In the lower catchment, lower rainfall and finer-gained sediments reduce the magnitude and variability of hydraulic gradient between the aquifer and the river, producing lower but more constant groundwater inflows. The water table in the lower reaches has a shallow gradient, and small changes in river height or groundwater level can result in fluctuating gaining and losing behaviour. The middle catchment represents a transition in river-aquifer interactions from the upper to the lower catchment. High baseflow in some parts of the middle and lower catchments is caused by groundwater flowing over basement highs. Mass balance calculations based on 222Rn activities indicate that groundwater inflows are 2 to 17% of total flow with higher inflows occurring during high flow periods. In comparison to 222Rn activities, estimates of groundwater inflows from Cl concentrations are higher by up to 2000% in the upper and middle catchment but lower by 50 to 100% in the lower catchment. The high baseflow estimates using Cl concentrations may be due to the lack of sufficient difference between groundwater and surface water Cl concentrations. Both hydrograph separation and differential flow gauging yield far higher baseflow fluxes than 222Rn activities and Cl concentrations, probably indicating the input of other sources to the river in additional to regional groundwater, such as bank return flows.