Borrelia burgdorferi sensu stricto (s.s.) was isolated from questing adult Ixodes scapularis Say ticks collected from Turkey Point Provincial Park (TPPP), Ontario, Canada during 2005–2006. DNA from ten (67%) of 15 pools of ticks was confirmed positive for B. burgdorferi s.s. using polymerase chain reaction (PCR) by targeting the rrf (5S)-rrl (23S) intergenic spacer region and OspA genes. This significant infection rate indicates an accelerated development of B. burgdorferi s.s. in TPPP, because this pathogen was not detected five years previously during sampling of the three motile life stages of I. scapularis. Our study provides the initial report of the presence of B. burgdorferi s.s. in TPPP, which is now endemic for Lyme disease. Ultimately, people and domestic animals are at risk of contracting Lyme disease when they frequent this park.