The genetic diversity of Borrelia burgdorferi isolates from several geographic regions was evaluated by nucleotide sequence analysis of the genes encoding 23S ribosomal RNA and outer surface protein A. Comparison of nucleotide sequences spanning 738 bp of the 23S ribosomal DNA from two unusual isolates, DN127 (Del Norte County, California) and 25015 (Millbrook, New York), to homologous sequences from other B. burgdorferi isolates from the United States and Russia identified several nucleotide sequence polymorphisms that are unique to these two isolates. Sequence analysis of a 615 nucleotide segment of the gene encoding outer surface protein A also revealed greater similarity of strains DN127 and 25015 (94.1%) compared to other US and Eurasian isolates. These data were further corroborated by genomic macrorestriction analysis, in which DN127 and 25015 demonstrated unique restriction digestion patterns. Our findings suggest that substantial genetic diversity of B. burgdorferi, rivaling that of European strains, exists among isolates from the United States. Strains DN127 and 25015 are unique among all B. burgdorferi isolates tested to date, and though isolated from opposite longitudinal extremes of the North American continent, are closely related.