The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%; CI 95%: 0.1–3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.