The first local spread of COVID-19 in Israel was detected in March 2020. Due to the diversity in clinical presentations of COVID-19, diagnosis by RT-PCR alone might miss patients with mild or no symptoms. Serology testing may better evaluate the actual magnitude of the spread of infection in the population. This is the first nationwide seroprevalence study conducted in Israel. It is one of the most widespread to be conducted thus far, and the largest per-country population size. The survey was conducted between June 28 and September 14, 2020 and included 54,357 patients who arrived at the Health Maintenance Organizations to undergo a blood test for any reason. A patient was considered seropositive after two consecutive positive results with two different kits (Abbott and DiaSorin).The overall seroprevalence was 3.8% (95%CI 3.7–4.0), males higher than females [4.9% (95%CI 4.6–5.2) vs. 3.1% (95%CI 2.9–3.3) respectively]. Adolescents had the highest prevalence [7.8% (95%CI 7.0–8.6)] compared to other age groups. Participants who had undergone RT-PCR testing had a tenfold higher risk to be seropositive. The prevalence-to-incidence ratio was 4.5–15.7. Serology testing is an important complimentary tool for assessing the actual magnitude of infection and thus essential for implementing policy measures to control the pandemic. A positive serology test result was recently accepted in Israel as being sufficient to define recovery, with possible far-reaching consequences, such as the deploying of employees to ensure the maintenance of a functional economy.