Summary It is well established that a river’s natural flow regime is a key determinant of ecological integrity and that dam regulated-flow releases can be detrimental to biotic communities and even affect river ecosystem structure (e.g. Poff and Zimmerman, 2010). Regional flow classes, groups of rivers that share similar natural flow regimes (called ‘river types’ by Poff and Zimmerman (2010)) and to which regional fish communities are ‘adapted’, have been proposed as units of analysis to identify significant damming related flow alteration (e.g. Poff, 1996; Poff and Zimmerman, 2010; McManamay et al., 2012a). Specifically, the natural range of flow behaviour within regional classes can be used to identify clearly anomalous flow features in rivers regulated by dams. Through ordination analysis on 70 ecologically important flow indices, we isolated five distinctive regional groupings of natural flow regimes among the 96 unregulated rivers located in study regions of South Eastern and South Western Canada, selected based on watershed characteristics as possible references for the 13 hydro-regulated, NSERC-HydroNet study rivers in British Columbia, Alberta, Ontario, Quebec and New Brunswick. The distinguishing characteristics of natural flow regimes within each flow class are explored through visualization in principal component space. The 16 regulated HydroNet sites were assigned to appropriate regional flow classes through discriminant function analysis based on shared geographic location and watershed characteristics. Anomalous flow features in the regulated rivers are then characterized by type and strength, based on identification of flow indices that are significantly different from observed natural variability in the relevant regional class. The magnitude distributions and the main axes of variability in index anomalies are analysed, across regions and regulation types (storage, peaking and run-of-the-river (RoR)). We also discuss the potential biological implications of the dominant flow anomalies in this set of Canadian rivers regulated by hydro dams.