A total of 305 ticks (21 larvae, 243 nymphs, 19 females and 22 males) were collected by flagging of vegetation in suburban woods of Pisarky Park (city of Brno) from July to October 2002. The midgut of each tick was dissected out and transferred individually into BSK-H medium. After cultivation, all specimens were examined by dark-field microscopy (DFM) for the presence of borreliae. Out of 305 tick samples, 45 were (14.8%) DFM positive. The following polymerase chain reaction (PCR) then revealed 37 (12.1%) samples positive for the presence of Borrelia burgdorferi sensu lato DNA. All 37 samples were further analysed by restriction fragment length polymorphism (RFLP) method. PCR-RFLP analysis revealed 14 strains of B. afzelii (37.8%), 15 strains of B. garinii (40.5%) and 2 strains of B. burgdorferi sensu stricto (5.4%). Four samples (10.8%) showed a mixed population of these genospecies. Two samples produced atypical RFLP pattern which were detected by sequence analysis as B. valaisiana (5.4%). Isolation attempts resulted in 21 spirochaetal strains (including two stains of B. valaisiana). The results show the diversity of B. burgdorferi s.l. in tick population and refer the risk of infection by pathogenic borreliae in Brno.