Regulation of river flow influences fish assemblages by changing availability of habitat and food sources and altering water quality parameters such as temperature and dissolved oxygen. These impacts of flow regulation on fish assemblages are often not observed until well after flow regulation has begun. Consequently, fish assemblages in rivers with a short-term history of flow regulation may not show obvious changes associated with flow regime change compared with rivers with longer periods of regulation. Environmental flow programs are designed to conserve and restore important elements of the natural flow regime needed to sustain fish assemblages and aquatic ecosystems in rivers with regulated flow regimes. The use of such flow restoration projects as ecological experiments is considered the best way to understand ecosystems at the scales of interest for environmental managers. This study examined the association of fish assemblage composition with flow regime in two regulated and two unregulated tributaries of the Hunter River, coastal New South Wales, Australia between 2006-2007.