During spring 2006–2009, eggs were collected for analysis of total thiamine from gravid rainbow smelt (Osmerus mordax) captured in each of the Great Lakes and two other waters as references for comparison. Mean standard length (mm ± standard error) of gravid females significantly differed between sample waters, with the Atlantic Ocean population being the longest (189 ± 12.3 mm) and Lake Michigan population the shortest (122 ± 0.3 mm). Mean thiamine concentrations (nmol/g ± standard error) for single-year samples for Lake Huron, Lake Michigan, and Little Clear Pond (New York) were 9.9 ± 0.8, 3.9 ± 0.7, and 8.1 ± 2.3 nmol/g, respectively. Thiamine concentrations for multiple-year samples ranged from 1.1 to 15.6 for Lake Ontario, from 2.6 to 3.3 for Lake Erie, from 5.0 to 9.9 for Lake Superior, and from 10.9 to 13.3 for the Atlantic Ocean (Fore River). Although highly variable within populations and across years, thiamine concentrations in most spawning adults appeared to be adequate in all the waters for the years sampled except for 2006 and 2009 in Lake Ontario and 2009 in Lake Erie.