The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, was discovered in blacklegged ticks, Ixodes scapularis Say at Turkey Point, Ontario, Canada. We report the first isolation of B. burgdorferi from a vertebrate animal collected on mainland Ontario. During this 2-yr study, spirochetes were isolated from the white-footed mouse, Peromyscus leucopus Rafinesque, and attached I. scapularis larvae. Similarly, isolates of B. burgdorferi were cultured from blacklegged tick adults, and confirmed positive with polymerase chain reaction by targeting OspA and rrf (5S)-rrl (23S) genes. Moreover, all isolates of B. burgdorferi from this area had complementary genetic structure, and the second primer set amplicons confirmed the first primer set amplification products. These findings show an epicenter endemic for B. burgdorferi within an established population of I. scapularis at Turkey Point.