Thiamine Deficiency Complex (TDC) limits early life stage survival of salmonines. Consuming fatty prey has been hypothesized as a cause of thiamine deficiency; however, this relationship has not been evaluated in the Laurentian Great Lakes where TDC occurs. We found that alewife (Alosa pseudoharengus) have higher lipid content than other common Lake Ontario prey fish. In addition, alewife were predicted as the most consumed prey for brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), lake trout (Salvelinus namaycush), and steelhead trout (O. mykiss); however, the relative importance of alewife in diet composition varied within and among species. Overall, species with greater predicted consumption of alewife had lower egg and muscle thiamine concentrations. Negative correlations between thiamine concentrations and both lipid content and fatty acid concentrations (mg/mg of wet tissue) were limited to brown trout. Similarly, negative correlations between fatty acid proportions (i.e., cumulative proportions of polyunsaturated fatty acids [PUFA] and monounsaturated fatty acids [MUFA]) and thiamine concentrations were only observed for brown and lake trout. Combining data from all species produced curvilinear correlations between thiamine concentrations (egg and muscle) and fatty acid composition (eggs and belly flap). Proportions of PUFAs had negative correlations with thiamine concentrations while proportions of MUFAs had positive correlations. These results provide evidence that, in some cases, salmonine fatty acid composition negatively correlates with thiamine concentrations in Lake Ontario; however, additional research is needed to confirm that this mechanism causes TDC in salmonines, and to understand additional factors potentially associated with TDC.