The coccidian parasite Toxoplasma gondii is found worldwide infecting warm-blooded vertebrates. Felids are the definitive hosts; other species act as intermediate hosts. Squirrels (Sciuridae) generally have high population densities in cities and forage and cache food on the ground, where they may come into contact with T. gondii oocysts or be preyed upon by cats and other carnivores. This environment might make squirrels important intermediate hosts of T. gondii in cities, and infection rates could indicate environmental levels of oocysts in soil. We investigated whether urban squirrels would be more exposed to T. gondii infection than rural squirrels with samples collected from American red squirrels (Tamiasciurus hudsonicus), eastern grey squirrels (Sciurus carolinensis), northern flying squirrels (Glaucomys sabrinus), and least chipmunks (Tamias minimus) in and around Winnipeg, Manitoba, Canada. We tested 230 tissue samples from 46 squirrels for T. gondii DNA by quantitative PCR and 13 serum samples from grey squirrels for T. gondii antibodies by competitive ELISA. We found no evidence of infection in any squirrel, indicating that squirrels are probably not important intermediate hosts of T. gondii in cities and that consumption of oocysts in the soil in general may not be an important contributor to transmission in colder environments.