Toxoplasmosis in wild turkeys (Meleagris gallopavo) is of epidemiological interest because turkeys feed from the ground, and detection of infection in turkeys indicates contamination by oocysts in the environment. During the 2018 spring hunting season in Pennsylvania, fresh (unfixed, not frozen) samples were obtained from 20 harvested wild turkeys and tested for Toxoplasma gondii infection. Hearts from all wild turkeys and skeletal muscle from 1 were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Antibodies to T. gondii were detected in 1:5 dilution of neat serum from 5 of 15 wild turkeys and in fluid from the heart of 1 of 4 wild turkeys with the modified agglutination test (MAT); neat serum was not available from 4 wild turkeys. Viable T. gondii was isolated from hearts of 5 wild turkeys, 1 with MAT of 1:10, 1 with MAT of 1:5, and 3 seronegative (MAT < 1:5). Toxoplasma gondii was isolated from both heart and skeletal muscle in the 1 wild turkey that had skeletal muscle submitted. The KO mice inoculated with tissue from all 5 infected wild turkeys died or were euthanatized when ill, 7–21 days post-inoculation (PI). Tachyzoites were detected in lungs of all KO mice, and the T. gondii strains were successfully propagated in cell culture. The SW mice inoculated with tissues of wild turkeys remained asymptomatic, and tissue cysts were seen in the brains of infected mice when euthanatized in good health at 46 days PI; 1 of the 2 SW mice inoculated with the heart of 1 turkey died on day 26, and tachyzoites were detected in its lung. Genetic typing on DNA extracted from culture-derived tachyzoites using the PCR restriction fragment length polymorphism with 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed that 4 isolates belonged to ToxoDB PCR-RFLP genotype #5 and 1 was genotype #216.