Local ecological attributes of streams are known to have strong influences on community membership for many aquatic insects. Differences in aquatic insect assemblages, therefore, should be clearly detectable across large scale ecological “units”, such as ecoregions. Many studies of aquatic invertebrates however, have suffered from a lack of species level identifications. In addition, many previous studies that examined the influence of ecoregion on aquatic assemblages have the implicit assumption that members of different taxa are responding in the same manner. Our study, therefore, was restricted to an ecologically (lotic) and functionally (mostly filter-feeding) homogenous group, the Simuliidae. In the current study, we examine the relationship between species assemblages of preimaginal blackflies and the landscape through which their stream habitats flow. Accordingly, the larval simuliid faunas from South Carolina, USA, are compared among three ecoregions established a priori: Blue Ridge Mountains, Piedmont, and Sandhills. Using discriminant function analysis, we show that each ecoregion produces a distinct stream habitat; factors responsible for regionalization are quantified. We also show that streams can be assigned correctly to ecoregion of origin 85% of the time on the basis of the simuliid assemblage. We suggest that our results can be interpreted most readily by considering the distribution of individual species.