A suite of indicators are often proposed during the development phase of an environmental monitoring program because they may have the potential to be influenced by an environmental disturbance of interest (e.g., riparian clearing ). Gradient analysis on such indicators can be used to identify a subset of those indicators that respond predictably to environmental disturbance gradients. When assessing the ecological effects of flow alteration, one of the challenges when using a gradient analysis approach is that climatic, topographic, and land use characteristics can have an over-riding effect on flow, as well as, physicochemical and biological processes in rivers. As such, broad-scale drivers such as climate and topography must be accounted for so that more localised influences, such as changes to the hydrological regime, may be identified. Given these challenges, our goal was to evaluate the usefulness of a gradient analysis approach for selecting ecological indicators of altered flow regimes in the Murray-Darling Basin (MDB), Australia. More specifically, we assessed whether potential indicators respond predictably to flow disturbance gradients after accounting for climatic, topographic, and land-use effects, and identified which characteristics of flow disturbance were most strongly associated with the indicators . The results of these analyses showed that some indicators of fish health and reproductive success were impacted by flow disturbance after accounting for other natural and land-use influences. Hence, the gradient analysis approach used here shows promise as a tool for assessing the ecological effects of altered flow regimes at basin-wide scales.