Humans have infected a wide range of animals with SARS-CoV-2, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.