Spatial variability of aquatic macroinvertebrates was examined in riffles of second to fourth order streams in Onondaga Creek, in central New York, USA. Even with only small differences in stream sizes, aquatic macroinvertebrates were distributed primarily by a headwater-to-valley gradient as defined by mean stream width and water surface slope. Secondary and tertiary gradients were based on hydraulic character. Direct gradient analysis using canonical correspondence analysis (CCA) examined the common and rare (respectively, < 5—0.5% and 0.5—0.001% of total) macroinvertebrate assemblage among sites. The variables used in the analysis were mean wetted width, water surface slope, mean Froude number and Froude number variance. The first CCA axis explained 43.1% of the variability. Froude number variance and Froude number affected the second and third CCA axes most strongly. The gradients revealed by the second and third CCA axes did not influence lower gradient valley sites, but did separate the headwater sites based on hydraulic character. Taxa were also distributed by functional feeding groups (i.e., collector-gatherer). The headwater-to-valley gradient did separate taxa slightly by functional feeding groups; however, the hydraulic gradient clearly separated scrapers and shredders from collector filterers.