Raptors are good indicators of the prevalence of Toxoplasma gondii in the environment because they prey on small mammals and birds. These prey species are a major source of infection in domestic cats (Felis catus), which shed the environmentally resistant oocysts. We assessed T. gondii infection in 281 opportunistically available raptors at a rehabilitation facility between 2012 and 2014. Antibodies to T. gondii were assayed by a modified agglutination test (cutoff 1:25) and found in serum of 22/71 Red-tailed Hawks (Buteo jamaicensis), 25/54 Barred Owls (Strix varia), 9/41 Red-shouldered Hawks (Buteo lineatus), 13/28 Great Horned Owls (Bubo virginianus), 6/20 Broad-winged Hawks (Buteo platypterus), 2/16 Eastern Screech Owls (Megascops asio), 12/13 Bald Eagles (Haliaeetus leucocephalus), 6/12 Cooper's Hawks (Accipiter cooperii), 1/8 Black Vultures (Coragyps atratus), and 1/1 Golden Eagle (Aquila chrysaetos). Antibodies were not detected in 5 Barn Owls (Tyto alba), 3 American Kestrels (Falco sparverius), 1 Mississippi Kite (Ictinia mississippiensis), and 1 Osprey (Pandion haliaetus). Viable T. gondii was isolated from the tissues of 1 antibody-positive Barred Owl and identified as a strain having type II alleles at all 10 loci tested, except one (ToxoDB polymerase chain reaction–restriction fragment length polymorphism genotype 3). Type II strain is the most common strain in the US. Results of this study indicate a high prevalence of T. gondii in some raptor species and the first reported genotyping from a Barred Owl.