Unpredictable recruitment and physical abnormalities (sores and lesions) have been observed in populations of Micropterus dolomieu (Smallmouth Bass) throughout the Susquehanna River basin. Malnutrition has been proposed as one of among several potential stressors, yet little to no information was available to critically assess its feasibility as a causal factor. We measured thiamine profiles of Smallmouth Bass (free thiamine [T], thiamine monophosphate [TP], and thiamine pyrophosphate [TPP]) for 3 tissues (egg, liver, and muscle) collected at 13 sites in the Susquehanna River and compared the values to those in 2 neighboring drainages (Allegheny River and Delaware River). Mass-specific thiamine concentrations in eggs were comparable to published values for Micropterus salmoides (Largemouth Bass), but higher than those found in Sander vitreus (Walleye), and Salvelinus namaycush (Lake Trout) known to consume Alosa pseudoharengus (Alewife), a thiaminase positive forage fish. In general, Smallmouth Bass collected from sites within the Susquehanna River basin had thiamine concentrations comparable to fish at the site in the Allegheny River, yet average thiamine concentrations in fish from the Susquehanna and Allegheny sites were each considerably lower than the average value collected from the Smallmouth Bass in the Delaware River. Future studies should consider a more balanced sampling design among watersheds to assess spatial variability among sites and basins. Average site-specific thiamine concentrations measured in Smallmouth Bass exceeded published minimum threshold values for Lake Trout. Given that Smallmouth Bass appear to have distinct thiamine profiles, concentrations, and timing of egg development, threshold thiamine concentrations parameterized for salmonids may not apply to Smallmouth Bass. As such, empirical studies that parameterize species-specific thiamine thresholds are needed to formally evaluate if thiamine deficiency is an issue for Smallmouth Bass in the Susquehanna River basin. To our knowledge, these are the first data on thiamine concentrations published for Smallmouth Bass.