Stream fish assmemblage data for 34 sites in Wisconsin and Minnesota were obtained from archived sources and were used in conjunction with long—term hydrological data to test the hypothesis that functional organization of fish communities is related to hydrological variability. For each of the 106 species present in the data set, six categories of species traits were derived to describe habitat, trophic, morphologica, and tolerance characteristics. A hierarchical clustering routine was used to identify two functionally similar groups of assemblages defined in terms of species presence/absence. Hydrological factors describing streamflow variability and predictability, as well as frequency and predictability of high flow and low flow extremes, were derived for each of the 34 sites and employed to explain differences among the functionally defined groups. Canonical discriminant analysis revealed that the hydrological data could clearly separate the two ecologically defined groups of assemblages, which were associated with either hydrologically variable streams (high coefficient of variation of daily flows, moderate frequency of spates) or hydrologically stable streams (high predictability of daily flows, stable baseflow conditions). Discriminant functions based on hydrological information classified the 34 fish assemblages into the correct ecological group with 85% accuracy. Assemblages from hydrologically variable sites had generalized feeding strategies, were associated with silt and general substrata, were characterized by slow—velocity species with headwater affinities, and were tolerant to silt. Proportions of species traits present at the 34 sites were regressed against an index of hydrological stability derived from a principal components analysis to test the hypothesis that functional organization of assemblages varied across a gradient of hydrological stability. Results were complementary with the discriminant analysis. Findings were in general agreement with theoretical predictions that variable should support resource generalists while stable habitats should be characterized by a higher proportion of specialist species. Several species of fish were identified as indicative of the variable—stable hydrological gradient among stream sites. A taxonomic analysis showed strong geographic patterns in species composition of the 34 assemblages. However, zoogeographic constraints did not explain the observed relationship between stream hydrology and functional organization of fish assemblages. The strong hydrological—assemblage relations found in the 34 midwestern sites suggest that hydrological factors are significant environmental variables influencing fish assemblage structure, and that hydrological alterations induced by climate change (or other anthropogenic disturbances) could modify stream fish assemblages structure in this region.