The genetic polymorphism of Borrelia burgdorferi and Borrelia afzelii, two species that cause Lyme borreliosis, was estimated by sequence typing of four loci: the rrs-rrlA intergenic spacer (IGS) and the outer-membrane-protein gene p66 on the chromosome, and the outer-membrane-protein genes ospA and ospC on plasmids. The major sources of DNA for PCR amplification and sequencing were samples of the B. burgdorferi tick vector Ixodes scapularis, collected at a field site in an endemic region of the north-eastern United States, and the B. afzelii vector Ixodes ricinus, collected at a similar site in southern Sweden. The sequences were compared with those of reference strains and skin biopsy isolates, as well as database sequences. For B. burgdorferi, 10-13 alleles for each of the 4 loci, and a total of 9 distinct clonal lineages with linkage of all 4 loci, were found. For B. afzelii, 2 loci, ospC and IGS, were examined, and 11 IGS genotypes, 12 ospC alleles, and a total of 9 linkage groups were identified. The genetic variants of B. burgdorferi and B. afzelii among samples from the field sites accounted for the greater part of the genetic diversity previously reported from larger areas of the north-eastern United States and central and northern Europe. Although ospC alleles of both species had higher nucleotide diversity than other loci, the ospC locus showed evidence of intragenic recombination and was unsuitable for phylogenetic inference. In contrast, there was no detectable recombination at the IGS locus of B. burgdorferi. Moreover, beyond the signature nucleotides that specified 10 IGS genotypes, there were additional nucleotide polymorphisms that defined a total of 24 subtypes. Maximum-likelihood and parsimony cladograms of B. burgdorferi aligned IGS sequences revealed the subtype sequences to be terminal branches of clades, and the existence of at least three monophyletic lineages within B. burgdorferi. It is concluded that B. burgdorferi and B. afzelii have greater genetic diversity than had previously been estimated, and that the IGS locus alone is sufficient for strain typing and phylogenetic studies.