Twenty-nine European and North American Borrelia burgdorferi strains isolated from patients with Lyme borreliosis, were investigated by restriction fragment length polymorphism (RFLP) of two phylogenetically highly conserved chromosomal genes encoding flagellin (fla) and the p60 common antigen (CA), as well as of the plasmid-borne outer surface protein A (ospA) gene. RFLP of the ospA, fla and CA gene revealed five, two and four distinct subspecies-specific patterns, respectively. RFLP classification of the B. burgdorferi strains was compared with four different classification schemes proposed by others: (i) molecular mass profile of OspA and OspB (Adam et al. [1]); (ii) OspA serotyping (Wilske et al. [34]); (iii) genomic fingerprinting on the central region of the B. burgdorferi fla gene (Picken [24]) and (iv) 16S rRNA signature nucleotide analysis (Marconi and Garon [19]). Results obtained with the different methods correlated highly. All strains classified as B. burgdorferi sensu stricto and B. afzelii could be unequivocally identified as one distinct group by all five typing methods. B. garinii isolates, however, were more heterogeneous and according to RFLP of the CA and ospA gene fell into either two or three subgroups. The agreement of the different approaches supports the recent concept that B. burgdorferi sensu lato strains should be delineated to three genomic groups and that B. burgdorferi sensu lato is clonal. All 12 US strains were B. burgdorferi sensu stricto, whereas the 17 European isolates belonged to any of three genospecies. Among European B. burgdorferi isolates there was an association between B. burgforferi genospecies and the clinical manifestation of Lyme borreliosis. B. afzelii strains were found to predominate in 11 skin isolates (75%), whereas all 6 cerebrospinal fluid isolates from patients with neuroborreliosis were B. garinii. These findings support the concept of a strain-dependent organotropism of B. burgdorferi.