Abstract Borrelia miyamotoi disease is an emerging tick-borne human illness in the United States caused by Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) bacterium. With Pennsylvania reporting thousands of tick-borne disease cases annually, determining the minimum infection rate (MIR) of B. miyamotoi in Ixodes scapularis (Say, Acari: Ixodidae) adults within Pennsylvania is of utmost importance. Active surveillance was performed from October 2019 to April 2020 to collect a minimum of 50 I. scapularis ticks from every county within Pennsylvania and then screened for B. miyamotoi via qPCR. Ticks were collected from all 67 counties with the majority of those being adult I. scapularis. Additional ticks collected were Dermacentor albipictus (Packard, Acari: Ixodidae), Haemaphysalis longicornis (Neumann, Acari: Ixodidae), and immature I. scapularis. Adult I. scapularis were pooled and tested for B. miyamotoi. MIR for positive B. miyamotoi pools and density of infected adult I. scapularis varied by county, with positive pools from 38 Pennsylvania counties. This is the first statewide evaluation of B. miyamotoi in Pennsylvania in questing adult I. scapularis. These prevalence and distribution data will aid health care practitioners within the state of Pennsylvania and the northeast United States to understand potential risk and bring awareness to the lesser known human Borrelia illness, Borrelia miyamotoi disease.