Toxoplasma gondii infections are prevalent in most warm-blooded animals worldwide. During the 2018 November hunting season in Pennsylvania, fresh (unfixed, not frozen) samples obtained from 99 harvested elk (Cervus canadensis) were tested for T. gondii infection. Antibodies to T. gondii were detected in 69 of 99 (69.7%) elk tested by the modified agglutination test (MAT, 1:25 cut-off). Tongues and hearts from 16 elk with high MAT titers (>1:200) were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Viable T. gondii was isolated from tongues of 2 elk with MAT titers of 1:200 and 1:3,200. Toxoplasma gondii from both isolates were successfully propagated in cell culture. 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 both isolates belonged to ToxoDB PCR-RFLP genotype #5 that is widely prevalent in wildlife in the United States. Our results suggest that elk may clear T. gondii organisms from their tissues.