Knowledge of the dietary components of fish species is important for understanding their growth, survival, and recruitment. Deficiency in thiamine (vitamin B1) leading to reproductive failure and physiological illness among freshwater fishes has been attributed to thiaminase activity in fish in the Great Lakes and the New York Finger Lakes, but the causes of variation in thiaminase activity among freshwater fishes is unclear. We characterized thiaminase activity in 29 species of freshwater fishes across 7 ray-finned and 1 jawless family. All fish were further categorized by phylogeny, trophic category (trophic level and feeding mode), and native or non-native status to evaluate how ecological processes correspond with thiaminase activity. Thiaminase activity varied significantly across species, families, trophic factors, phylogenetic groups, and sites. Teleosts that were more recently derived had higher thiaminase activity than more basal species. Thiaminase activity was also higher among herbivores than omnivores or carnivores. This trend was clearest in the Cyprinidae family, which had the greatest range in thiaminase activity and a wide range in trophic-level position and trophic categories (herbivores, omnivores, and carnivores). Variation in average thiaminase activity of Spotfin Shiners (Cyprinella spiloptera) among sites within a watershed was correlated with anthropogenic and natural components of land cover. Our study contributes much-needed quantitative ecological information related to thiaminase activity in a suite of fish species that vary in evolutionary history, trophic level, and foraging modes. However, more studies are needed to identify interacting causes of thiaminase variation and examine the implications of these findings on the overall health of aquatic populations and freshwater ecosystems.