Streams are naturally hierarchical systems, and their biota are affected by factors effective at regional to local scales. However, there have been only a few attempts to quantify variation in ecological attributes across multiple spatial scales. We examined the variation in several macroinvertebrate metrics and environmental variables at three hierarchical scales (ecoregions, drainage systems, streams) in boreal headwater streams. In nested analyses of variance, significant spatial variability was observed for most of the macroinvertebrate metrics and environmental variables examined. For most metrics, ecoregions explained more variation than did drainage systems. There was, however, much variation attributable to residuals, suggesting high among-stream variation in macroinvertebrate assemblage characteristics. Nonmetric multidimensional scaling (NMDS) and multiresponse permutation procedure (MRPP) showed that assemblage composition differed significantly among both drainage systems and ecoregions. The associated R-statistics were, however, very low, indicating wide variation among sites within the defined landscape classifications. Regional delineations explained most of the variation in stream water chemistry, ecoregions being clearly more influential than drainage systems. For physical habitat characteristics, by contrast, the among-stream component was the major source of variation. Distinct differences attributable to stream size were observed for several metrics, especially total number of taxa and abundance of algae-scraping invertebrates. Although ecoregions clearly account for a considerable amount of variation in macroinvertebrate assemblage characteristics, we suggest that a three-tiered classification system (stratification through ecoregion and habitat type, followed by assemblage prediction within these ecologically meaningful units) will be needed for effective bioassessment of boreal running waters.