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1. Assessment of the performance of protected areas in conserving freshwater biodiversity has been limited, has mostly involved small-scale studies and has produced mixed findings. 2. I analysed monitoring data from Australia’s Murray-Darling Basin (>10(6)km(2) and mostly arid or semi-arid) to compare fish assemblages between rivers inside and outside of protected areas. 3. The average richness and abundance of native species were significantly lower at sites within protected areas, but these sites were weighted towards steeper terrain and colder climates. 4. When analysis was confined to a subset of geographically and environmentally matched sites, the richness and abundance of native species did not differ significantly between protected and unprotected areas, and only two native species were significantly more abundant within protected areas, whereas another two were significantly more abundant outside. 5. Synthesis and applications. Protected status probably has little effect on native fish in the Murray-Darling Basin because it does not, by itself, exclude threats such as alien fish and alteration of water regimes. My findings therefore support the view that reserves need to be designed and managed specifically for freshwater species if they are to be most effective in their conservation. In the Murray-Darling Basin, and in similar regions around the world, actions such as greater control of alien species and allocation of adequate environmental flows will likely be critical to enabling protected areas to realize their potential for aquatic conservation.