Lyme disease has continued to spread in the Czech Republic, and there have been increasing numbers of cases in central and eastern Bohemia and Moravia. We have shown that Borrelia burgdorferi sensu lato causes infection by migration through connective tissues of the skin, heart, liver, and synovium without inflammation and by adhesion to host cells. Twenty-three isolates of B. burgdorferi sensu lato originally cultured from cerebrospinal fluid, blood, and tissue samples were characterized phenotypically by immunosorbent electron microscopy and Western blots and genotypically by sequence analysis of the outer surface (Osp) A and C gene fragments. Comparison of genotyping of borreliae performed directly in patients and in Ixodes ricinus ticks collected in the same endemic regions with typing of cultures revealed that a large proportion of directly typed samples had more heterogenic Borrelia types or combined sequence variants. In addition, B. garinii OspA types 6 and 5 were in coinfection with B. valaisiana and rarely with B. burgdorferi, but OspA subtype 4 was not found in the mixure. More frequent and heterogenic direct proof of B. burgdorferi sensu lato in CSF and blood versus culture reflects either different behavior and exigencies of different genospecies or intergenospecies exchange caused by human and animal migration.