Classifications are typically specific to particular issues or areas, leading to patchworks of subjectively defined spatial units. Stream conservation is hindered by the lack of a universal habitat classification system and would benefit from an independent hydrology-guided spatial framework of units encompassing all aquatic habitats at multiple spatial scales within large regions. We present a system that explicitly separates the spatial framework from any particular classification developed from the framework. The framework was constructed from landscape variables that are hydrologically and biologically relevant, covered all space within the study area, and was nested hierarchically and spatially related at scales ranging from the stream reach to the entire region; classifications may be developed from any subset of the 9 basins, 107 watersheds, 459 subwatersheds, or 10,000s of valley segments or stream reaches. To illustrate the advantages of this approach, we developed a fish-guided classification generated from a framework for the Great Lakes region that produced a mosaic of habitat units which, when aggregated, formed larger patches of more general conditions at progressively broader spatial scales. We identified greater than 1,200 distinct fish habitat types at the valley segment scale, most of which were rare. Comparisons of biodiversity and species assemblages are easily examined at any scale. This system can identify and quantify habitat types, evaluate habitat quality for conservation and/or restoration, and assist managers and policymakers with prioritization of protection and restoration efforts. Similar spatial frameworks and habitat classifications can be developed for any organism in any riverine ecosystem.