Borrelia miyamotoi, a bacterium that causes relapsing fever, is found in ixodid ticks throughout the northern hemisphere. The first cases of human infection with B. miyamotoi were identified in 2011. In the eastern USA, blacklegged ticks (Ixodes scapularis) become infected by feeding on an infected vertebrate host, or through transovarial transmission. We surveyed B. miyamotoi prevalence in ticks within forested habitats in Dutchess County, New York, and identified possible reservoir hosts. To assess spatial variation in infection, we collected questing nymphal ticks at > 150 sites. To assess temporal variation in infection, we collected questing nymphs for 8 years at a single study site. We collected questing larval ticks from nine plots to estimate the amount of transovarial transmission. To evaluate potential reservoir hosts, we captured 14 species of mammal and bird hosts naturally infested with larval blacklegged ticks and held these hosts in the laboratory until ticks fed to repletion and molted to nymphs. We determined infection for all ticks using quantitative polymerase chain reaction. The overall infection prevalence of questing nymphal ticks across all sites was ~ 1%, but prevalence at individual sites was as high as 9.1%. We detected no significant increase in infection through time. Only 0.4% of questing larval ticks were infected. Ticks having fed as larvae from short-tailed shrews, red squirrels, and opossums tended to have higher infection prevalence than did ticks having fed on other hosts. Further studies of the role of hosts in transmission are warranted. The locally high prevalence of B. miyamotoi in the New York/New England landscape suggests the importance of vigilance by health practitioners and the public.