Eastern Long Island, New York, is one of the major foci of Lyme disease in the United States. As in almost all other parts of North America, Lyme disease in this region is caused by a single genomic species of spirochete, Borrelia burgdorferi sensu stricto. For three consecutive years, natural populations of Lyme Borrelia in this region were sampled and studied for gene flow among different locations, changes in population structure over time, and selective forces. The genetic diversity of Borrelia populations was measured at the outer surface protein A (ospA) locus using Cold Single-Stranded Conformation Polymorphism (Cold SSCP) analysis. The Borrelia populations were found to be highly polymorphic within any of thirteen local populations. Ewens-Watterson tests of neutrality revealed that the high level of genetic diversity within local Borrelia populations is maintained by balancing selection. Frequency-dependent selection for the different strains distinguished by the ospA alleles is likely the mechanism of the balancing selection. Allele frequency distributions of Borrelia populations were homogeneous across the region in any particular year, although different infection rates of local tick (Ixodes scapularis) populations suggested that the Borrelia populations were at least partially isolated. Since the allele frequency distribution changed over time, while remaining homogeneous over space, the nearly uniform allele frequency distribution across the region cannot be explained by recent geographic expansion from a single population. This uniform distribution across the region thus may be maintained by selection, or by a significant amount of migration or both. The genetic structure of B. burgdorferi sensu stricto also differed between spirochetes infecting nymphal ticks and those infecting adult ticks. Since larval and nymphal ticks have distinctly different host feeding preferences, host adaptation of spirochete populations is implied. This distinction and an animal study using chipmunks suggest that ticks infected by Borrelia as larvae may have high mortality in the wild. This study represents a genetic analysis of local populations of a bacterial species.