Lyme borreliosis (LB) represents the most common vector-borne zoonotic disease in the Northern Hemisphere. The infection is caused by the spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex which circulate between tick vectors and vertebrate reservoir hosts. The complex of Borrelia burgdorferi s.l. encompasses at least 12 species. Genetic variability within and between each species has a considerable impact on pathogenicity, clinical picture, diagnostic methods, transmission mechanisms and its ecology. The distribution of distinct genospecies varies with the different geographic area and over a time. In recent years, new molecular assays have been developed for direct detection and classification of different Borrelia strains. Profound studies of strain heterogeneity initiated a new approach to vaccine development and routine diagnosis of Lyme borreliosis in Europe. Although great progress has been made in characterization of the organism, the present knowledge of ecology and epidemiology of B. burgdorferi s.l. is still incomplete. Further information on the distribution of different Borrelia species and subspecies in their natural reservoir hosts and vectors is needed.