The Ecological Limits of Hydrologic Alteration (ELOHA) is a new framework designed to develop environmental flow prescriptions for many streams and rivers in a user-defined geographic region or jurisdiction. This study presents hydrologic classifications and comparisons of natural and altered flows in southeast Queensland, Australia, to support the ecological steps of a field trial of the ELOHA framework. We extended existing protocols for flow classification by assessing the stability of flow classes. Model-based clustering distinguished six Reference classes (based on modelled pre-development flow data) and five Historic classes (based on stream gauge data). The principal flow regime change was loss of some of the original (natural) flow diversity accompanied by the emergence of a perennial flow class in the Historic classification composed mostly of gauges with flow regimes influenced by dams. However, similarities between Reference and Historic classifications indicate that hydrologic changes in southeast Queensland have not totally obscured Reference (pre-development) characteristics. Duration of low flow spells has undergone the greatest absolute change from Reference values. Dams had substantial but variable impacts on downstream flow regimes. Each dam created a unique downstream flow signature, indicating that environmental flow guidance for each regulated river must be tailored to the particulars of flow alterations, the associated ecological impacts and the desired future ecological state of the aquatic ecosystem. Other stressors were implicated in flow regime change, highlighting the need to consider the potential influence of factors other than prominent water infrastructure on flow regime alterations and associated ecological responses.