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Eskers are complex geological formations shaped with a linear accumulation of sand and gravel under the glaciers during the last ice age and that provide crucial resources such as drinking water, sand/gravel, outdoor recreational sites, and productive forests. Surrounding sand and gravel and connection with the groundwater influences the physicochemical properties of lakes on esker which can benefit different biotic communities in the food web. The sustainable management of resources provided by eskers requires baseline ecological knowledge of these ecosystems. However, very little information exists about the ecology of freshwater ecosystems on eskers. This study uses a food web approach to identify the environmental variables, biological diversity, and indicator species associated with esker lakes to better understand their ecological functioning and biodiversity patterns to benefit their sustainable management and conservation. Fifty lakes were sampled in the Abitibi-Témiscamingue region (Canada), half on eskers and half on the surrounding boreal clay belt to include the most abundant lake ecosystems of the region. Physicochemical, environmental, and anthropogenic variables measured in the two lake types showed that esker lakes differed markedly from clay lakes. Nutrient concentrations, conductivity, and macrophyte cover were significantly lower in esker lakes than in clay lakes, whereas dissolved oxygen saturation and concentration showed the opposite trend. Three interconnected trophic levels of the esker lake food webs—waterbird, fish, and macroinvertebrate communities—were characterized for biological diversity and the associated species. We found a significantly lower Shannon diversity index for waterbirds (mean ± standard deviation; 0.7 ± 0.2), fish (0.4 ± 0.3), and a tendency for a lower value for macroinvertebrates (0.9 ± 0.3) in esker lakes than the clay lakes (1.1 ± 0.4, 0.9 ± 0.3, and 1.3 ± 0.5, respectively). Common goldeneye (Bucephala clangula) and Canada goose (Bucephala clangula) were associated significantly with esker lakes and identified as indicator species for esker lakes. In contrast, ring-necked duck (Aythya collaris) and hooded merganser (Lophodytes cucullatus) were associated significantly with clay lakes. Perlidae was similarly associated with esker lakes as an indicator for macroinvertebrates. Anthropogenic activities such as forest harvesting have altered the waterbird community, and recreational activities around the lakes have modified the fish and macroinvertebrate communities. We conclude that esker lakes differ from other regional lakes and are associated with specific environmental and biological variables and indicator species. The biological diversity in esker lakes is lower than that of clay lakes for all studied trophic levels of the food web, but these waterbodies provide preferential habitats for some species. This research provides the first baseline ecological information necessary to establish sustainable management and conservation strategies for this vulnerable ecosystem.