The blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), has a wide geographical distribution in Ontario, Canada, with a detected range extending at least as far north as the 50th parallel. Our data of 591 adult I. scapularis submissions collected from domestic animals (canines, felines, and equines) and humans during a 10-yr period (1993–2002) discloses a monthly questing activity in Ontario that peaks in May and October. The Lyme disease spirochete Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt & Brenner was detected in 12.9% of I. scapularis adults collected from domestic hosts with no history of out-of-province travel or exposure at a Lyme disease endemic area. Fifty-three isolates of B. burgdorferi were confirmed positive with polymerase chain reaction by targeting the rrf (5S)-rrl (23S) gene. Using DNA sequencing of the ribosomal species-specific rrf (5S)-rrl (23S) intergenic spacer region, all isolates belong to the pathogenic genospecies B. burgdorferi sensu stricto (s.s.). Nucleotide sequence analysis of a 218- to 220-bp amplicon fragment exhibits six cluster patterns and, collectively, these isolates branch into four phylogenetic cluster groups for both untraveled, mammalian hosts and those with travel to the northeastern United States (New Jersey and New York). Four of five geographic regions in Ontario had strain variants consisting of three different genomic cluster groups. Overall, our molecular characterization of B. burgdorferi s.s. shows genetic heterogeneity within Ontario and displays a connecting link to common strains from Lyme disease endemic areas in the northeastern United States. Moreover, our findings of B. burgdorferi in I. scapularis reveal that people and domestic animals may be exposed to Lyme disease vector ticks, which have wide-ranging distribution in eastern and central Canada.