Evidence pointing to thiamine deficiency as a primary factor in early mortality syndrome in feral salmonids from the Great Lakes and New York's Finger Lakes continues to mount. Such deficiency is believed to be the result of the consumption of nontraditional forage fish, such as alewife Alosa pseudoharengus, that contain thiaminase activity. The source of thiaminase within these forage fish has not been reported. In this study, we report finding thiaminase-positive Paenibacillus thiaminolyticus and other Paenibacillaceae that are closely related to P. thiaminolyticus in the viscera of frozen alewives. Thiaminase-positive bacteria associated with alewife viscera have not previously been reported. Because viable thiaminase-positive cultures were found in only 25% of the fish, bacteria should be considered as only one potential source of thiaminase in alewives.