In this study the seroprevalence of anti-Borrelia burgdorferi IgG antibodies in populations of naturally exposed dogs in the Belgrade area was determined. Serum samples of 215 dogs were tested with in-house made indirect immunofluorescent-antibody assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blot assay (WB). Sera were from 34 pet dogs suspected of having Lyme borreliosis, 41 hunting dogs, 125 shelter dogs, and 15 clinically healthy pet dogs. Results of WB were used as a standard against which the performances of ELISA and IFA were evaluated. ELISA was significantly more sensitive than IFA (84.8 vs 67.9%), whereas the specificities of these tests were just slightly different (94.4% vs 92.6 %). Borrelia burgdorferi seroprevalence of 24.7% among tested dogs was recorded by WB test. The highest seroprevalence of 31.2% was found in the group of shelter dogs, while hunting and pet dogs showed lower seroprevalence of 19.5% and 17.6%, but the difference was statistically insignificant. These results are the first report on the seroprevalence of IgG antibodies to B. burgdorferi in dogs in the Belgrade area and could be of importance in the assessment of the risk of infection with B. burgdorferi in human population.