To determine whether the genospecies composition of Lyme disease spirochetes is spatially stratified, we collected questing Ixodes ricinus ticks in neighboring plots where rodents, birds, and lizards were present as reservoir host and compared the prevalence of various genospecies. The overall prevalence of spirochetes in questing ticks varied across the study site. Borrelia lusitaniae appeared to infect adult ticks in one plot at the same frequency as did Borrelia afzelii in the other plots. The relative density of questing nymphal and adult ticks varied profoundly. Where lizards were exceedingly abundant, these vertebrates seemed to constitute the dominant host for nymphal ticks, contributing the majority of infected adult ticks. Because lizards support solely B. lusitaniae and appear to exclude other genospecies, their narrow genospecies association results in predominance of B. lusitaniae in sites where lizards are abundant, while limiting its spread to the host's habitat range. To the extent that Central European B. lusitaniae strains are nonpathogenic, the presence of numerous lizards should locally decrease risk of infection for people. Evaluation of regional risk of infection by Lyme disease spirochetes should take the spatial effect of hosts into consideration, which stratify the distribution of specifically infected ticks on a small scale.