Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has been found to infect various domestic and wild animal species. In this study, convenience serum samples from 575 bison, 180 elk, and 147 samples from various wildlife species collected between 2020 and 2023 from several regions in the United States were analyzed for the presence of SARS-CoV-2-specific antibodies. Two commercial ELISA assays based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) or the nucleocapsid protein (N-ELISA) of SARS-CoV-2 were used. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT). Our results indicated that 1.2% of bison, 2.2% of elk, and 4.1% of the other wildlife species serum samples were seropositive in the sVNT, whereas 4.2% of bison, 3.3% of elk, and 1.4% of the other captive wildlife species serum samples tested positive by the N-ELISA. Among the sVNT serum samples, two samples from bison, one sample from elk, and five serum samples from other wildlife species (one cheetah, one gorilla, two lions, and one hippopotamus) had neutralizing antibody titers in the VNT, indicating these species are susceptible to SARS-CoV-2 infection. These findings highlight the importance of broad surveillance efforts for the effective monitoring of SARS-CoV-2 in non-human hosts.