Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a widespread problem with major implications for salmonid fish species. The mechanisms by which the bacterium has reached high levels of infection previously unrecorded in the Laurentian Great Lakes are presently unknown. Research involving reservoirs and mechanisms of R. salmoninarum transmission in fish is lacking because of the ecologic complexity of heterogeneous habitats and the lack of adequate funding. Herein, we report on the isolation of R. salmoninarum from the kidneys of the sea lamprey (Petromyzon marinus). The bacterium was cultured from kidneys of 16% and 4% of lampreys collected from two locations within the Lake Ontario watershed in 2003 and 2004, respectively. The identity of bacterial colonies was verified with the nested polymerase chain reaction and quantitative enzyme-linked immunosorbent assay.