Clinical and tick isolates of Borrelia burgdorferi sensu stricto, the bacterial agent of Lyme disease, from the northeastern United States were sequenced at 12 loci located on the main chromosome and 7 plasmids (lp54, cp26, cp9, lp17, lp25, lp28-2, and lp38). The outer surface protein C gene (ospC) showed the highest number (12) of major alleles (defined as alleles differing by 5% or more in nucleotide sequence) while other ORFs had only two to four major alleles. A non-recombining chromosomal marker, the rrs-rrlA ribosomal RNA spacer, was used to infer the intraspecific phylogeny among these B. burgdorferi isolates. We were thus able to analyze the multilocus genotypes in the context of a B. burgdorferi intraspecific phylogeny. Except for ospC, sequence variations at plasmid-borne loci showed broad inconsistency with the intraspecific phylogeny, supporting DNA exchanges mediated by plasmid transfers. The multilocus linkage frequently observed in B. burgdorferi populations is due more likely to a "founder effect" than to a lack of recombination. The exceptional phylogenetic consistency of ospC, in conjunction with its selectively maintained high intraspecific diversity, suggested a dominant role ospC plays in the initiation and maintenance of adaptive differentiation in B. burgdorferi.