To determine the seroprevalence of Lyme disease in gray wolves (Canis lupus) from various counties of Minnesota and Wisconsin (USA), 589 serum samples were collected from 528 wolves from 1972 to 1989. An indirect fluorescent antibody (IFA) test was used to detect the presence of antibodies against Borrelia burgdorferi. Titers of ≥1:100 were considered positive. Results were confirmed by testing a few selected sera by Western blotting. Of the 589 sera tested, 15 (3%) had IFA titers of ≥1:100. Three of the positive samples were collected from Douglas County in Wisconsin and twelve were from Minnesota counties. This study indicates that wolves are exposed to B. burgdorferi and are susceptible to Lyme disease.