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Black bears (Ursus americanus) are commonly exposed to Toxoplasma gondii. However, there are no reports of exposure or infection with T. gondii in black bears from Oklahoma. The purpose of our project was to determine the seroprevalence of T. gondii antibodies in black bears collected in Oklahoma. Additionally, since only serum was available from these bears, we sought to determine if DNA extraction and PCR amplification for T. gondii was possible on serum samples from bears with positive titers. Seroprevalence was determined using modified agglutination test (MAT). Serum was collected from 44 live-trapped bears in southeastern Oklahoma; 32 (73% ± 58–84%) had antibodies against T. gondii. Seroprevalence in adult bears (85% ± 67–95%) was significantly higher (p = 0.028) than yearlings (33.0% ± 56–80%). Adult bears were 3.4 times more likely to have antibodies to T. gondii than yearlings. From the bears with positive titers, T. gondii DNA was detected in 12 of the 32 seropositive samples by PCR of the B1 gene, with two of the samples showing variation in two nucleotide positions when compared with available sequences. Multilocus PCR-RFLP genotyping of these 12 samples revealed three ToxoDB genotypes, including #2 (type III, haplogroup 3), #4 (type XII, haplogroup 12), and #74 (haplogroup 12). To the best of our knowledge, this is the first report of T. gondii seroprevalence in black bears from Oklahoma. Our results indicate that exposure and infection with T. gondii in black bears from Oklahoma is common. [ABSTRACT FROM AUTHOR]