Viable Toxoplasma gondii was isolated by bioassay in mice from tissues of 2 feral cats (Felis domesticus), 2 raccoons (Procyon lotor), a skunk (Mephitis mephitis) trapped in remote locations in Manitoba, Canada, and a black bear (Ursus americanus) from Kuujjuaq, northern Quebec, Canada. Genotyping of these T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico revealed 4 genotypes. None of the isolates was clonal archetypal Types I, II, and III found in the United States. These results are in contrast with the Type II genotype that is widespread in domestic animals and humans throughout the United States and Europe. This is the first genotyping of T. gondii isolates from this part of North America.