Lyme borreliosis is very important for dermatovenereologists because of its skin manifestations that include erythema chronicum migrans, lymphadenosis benigna cutis, and aerodermatitis chronica atrophicans. In Croatia, the spirochete Borrelia burgodorferi was first isolated in 1991 at University Department of Dermatology and Venereology, Zagreb University Hospital Center. In October 2003, the Laboratory of Clinical Immunology, Immunofluorescence and Serodiagnosis was of the Ministry of Health and Social Welfare of the Republic of Croatia appointed Reference Center for the Diagnosis of Syphilis and Lyme Disease. Results of immunofluorescence diagnosis of Lyme disease are reported and areas in Croatia endemic for Lyme borreliosis are presented.