The conservation, management and monitoring of aquatic resources should benefit from understanding their spatial structuring. In this paper, we used the reference condition approach (RCA) to test if the variability in biotic communities, riverine fish assemblages in the present case, is better controlled with a spatial delineation based on ecoregions or by grouping rivers with local catchment characteristics. Electrofishing data from 493 riffles in 99 rivers in Finland were used in the analysis. We completed multivariate analyses (nonmetric multidimensional scaling, multiresponse permutation procedures) to identify differences in fish community composition among groups based on ecoregions or catchment characteristics. We concluded that both regional processes and local environmental characteristics structured fish populations, and bioassessment of freshwaters should be based on regional spatial delineations, supported by local scale characteristics. Spatial delineations like ecoregions should be used to assist the bioassessment to improve the efficiency to detect the human impacts.