The composition and structure of fish communities are affected by a variety of factors, both within the aquatic ecosystem and from the surrounding watershed. Many studies have examined what structures fish assemblages over broad spatial and environmental gradients. However, the influence of local environmental attributes on the observed variation in fish assemblages is less understood across finer spatial scales, where broad-scale climatic and anthropogenic factors are relatively similar. We used multiple linear regression to examine the relationships between environmental variables and various aspects of fish assemblages (including trophic function, community indices, and species composition) in 90 glacial lakes from northern Indiana, USA, from 1990 to 2010. Trophic structure and species composition were primarily related to water quality, whereas trophic level increased and omnivores declined as Secchi depth increased and phosphorus concentrations decreased. Species richness and diversity, in contrast, were positively linked to lake size and depth. We also found unique relationships among fish assemblages and environmental variables between samples collected using gill nets and night electrofishing, which may result from these gears sampling different assemblage components – therefore, relationships that were apparent in both sampling techniques (e.g., Secchi depth effects on trophic structure) may be the most robust and useful for improving aquatic ecosystem management on local scales.