Thiamine (vitamin B₁) deficiency in the Great Lakes has been attributed to elevated levels of thiaminase I enzyme activity in invasive prey species; however, few studies have investigated thiaminase activity in native prey species. Some of the highest levels of thiaminase activity have been measured in invasive dreissenid mussels with little understanding of background levels contributed by native freshwater mussels (Bivalvia: Unionidae). In this study, thiaminase activity was measured in two freshwater mussel species, Elliptio complanata and Strophitus undulatus, from the Delaware and Susquehanna River drainage basins located in north eastern United States. Thiaminase activity was also measured in gravid and non-gravid S. undulatus. Average thiaminase activity differed significantly between species (7.2 and 42.4 μmol/g/min, for E. complanata and S. undulatus respectively) with no differences observed between drainage basins. Gravid S. undulatus had significantly lower thiaminase activity (28.0 μmol/g/min) than non-gravid mussels (42.4 μmol/g/min). Our results suggest that a suite of factors may regulate thiaminase activity in freshwater mussels and that native freshwater mussel thiaminase activity is within the range observed for invasive dreissenids. These results add to our understanding of the complexities in identifying the ecological conditions that set the stage for thiamine deficiency.