We conducted a survey of biotic and abiotic factors correlated with waterbird assemblage composition in the Aspen Parkland, an endangered transitional zone between prairies and boreal forest, rich in waterbirds but poor in information relating avian distributions to features of aquatic habitats. We surveyed breeding aquatic bird species, plus invertebrates, fishes and limnological factors on 25 naturally eutrophic to hypertrophic lakes in Alberta, Canada. We observed 43 non-passerine species. Canonical Correspondence Analysis revealed two assemblage types: (a) a “grebe-gull” assemblage of gulls, grebes and waterfowl on hypertrophic lakes with small-bodied fishes and invertebrate assemblages of Diptera, Cladocera, Amphipoda and Glossiphoniidae, or (b) a “teal-shorebird” assemblage of ducks and shorebirds on eutrophic fishless lakes with invertebrate assemblages of Gastropoda, Erpobdellidae, Dytiscidae and Trichoptera. Lakes with “grebe-gull” assemblages were deeper and the presence of fish added to the resource base of these systems, perhaps contributing to greater species richness in “grebe-gull” lakes (x=12.5 species in presence of fishes, x=8.1 species in absence of fishes). Phytoplankton (chlorophyll-a) and nitrogen (TDN) concentrations were the most important limnological factors correlated with bird-assemblage composition, and may influence composition of invertebrate resources for birds on the two types of lakes. Our multivariate approach could be useful to managers for identifying key environmental factors associated with the successful management of multiple waterbird species in the Aspen Parkland.