Lyme disease is a zoonosis caused by various species belonging to the Borrelia burgdorferi bacterial species complex. These pathogens are transmitted by ticks and infect multiple, taxonomically distinct, host species. From an epidemiological perspective, it is important to determine whether genetic variants within the species complex are able to spread freely through the whole host community or, instead, if certain variants are restricted to particular hosts. To this end, we characterized the genotypes of members of the B. burgdorferi species complex; the bacteria were isolated from more than two hundred individuals captured in the wild and belonging to three different rodent host species. For each individual, we used a high-throughput approach to amplify and sequence rplB, a housekeeping gene, and ospC, which is involved in infection. This approach allowed us to evaluate the genetic diversity both within and among species in the B. burgdorferi species complex. Strong evidence of genetic differentiation among host species was revealed by both genes, even though they are, a priori, not constrained by the same selective pressures. These data are discussed in the context of the advancements made possible by multi-locus high-throughput sequencing and current knowledge of Lyme disease epidemiology.