For the past six decades, parasitic sea lampreys (Petromyzon marinus) have caused devastating losses to salmonid fisheries in the Great Lakes. To reduce the number of sea lampreys, the Great Lakes Fishery Commission began a large-scale program based on trapping male sea lampreys, sterilizing them, and releasing sterile males back into streams to compete with fertile males for spawning females. The transfer of lampreys among lakes can potentially lead to the transfer of various pathogens, and this has raised major concerns regarding the possibility of resident fish populations becoming infected by introduced pathogens. During a health inspection of sea lampreys collected from Lake Ontario, lampreys with obvious furuncle-like lesions (1–2 cm in diameter) were noticed. Most of the furuncles occupied the dorso-lateral musculature, and Aeromonas salmonicida subsp. salmonicida was isolated from the kidneys. This bacterium was cultured from kidneys of 2.5% of the sea lampreys collected from two locations within the Lake Ontario watershed in 2004. The identity of bacterial colonies was presumptively verified with biochemical reactions and confirmed with polymerase chain reaction. This is the first report of A. salmonicida infection in sea lamprey in the Great Lakes basin associated with furunculosis.