Simple SummaryCOVID-19 emerged in China in 2019. It is caused by an until-then unknown coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2). Through experimental infections in the search for a suitable animal model and reported infections in pets early in the pandemic, it became clear that several animal species may be susceptible to SARS-CoV-2 infection. According to the open access dataset of reported SARS-CoV-2 events in animals, about 119 zoo animals have been reported with a SARS-CoV-2 infection. However, the detection of SARS-CoV-2 infections in zoo animals has relied on the observation of symptoms (cough, nasal discharge), behaviour changes (reduced appetite, lethargy), or death of these captive animals. SARS-CoV-2 infections may therefore remain undetected if animals do not show obvious symptoms. In this study, we investigated the potential circulation of SARS-CoV-2 in zoo mammal species by sampling and screening faecal samples from all the mammals in two zoos in Belgium between September 2020 and July 2021 using molecular biology techniques. This study is the first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in all mammal species in a zoo. We conclude that at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.AbstractThe COVID-19 pandemic has led to millions of human infections and deaths worldwide. Several other mammal species are also susceptible to SARS-CoV-2, and multiple instances of transmission from humans to pets, farmed mink, wildlife and zoo animals have been recorded. We conducted a systematic surveillance of SARS-CoV-2 in all mammal species in two zoos in Belgium between September and December 2020 and July 2021, in four sessions, and a targeted surveillance of selected mammal enclosures following SARS-CoV-2 infection in hippopotamuses in December 2021. A total of 1523 faecal samples from 103 mammal species were tested for SARS-CoV-2 via real-time PCR. None of the samples tested positive for SARS-CoV-2. Additional surrogate virus neutralisation tests conducted on 50 routinely collected serum samples from 26 mammal species were all negative. This study is the first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in all mammal species of a zoo. We conclude that at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.