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Background The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and to calculate the infection fatality rate (IFR). These measures may help the authorities make informed decisions and adjust the current societal interventions. The objective was to perform nationwide real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population-based IFR. Methods Danish blood donors aged 17–69 years giving blood 6 April to 3 May were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between geographical areas, and an estimate of the IFR was calculated. Seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CIs). Results The first 20 640 blood donors were tested, and a combined adjusted seroprevalence of 1.9% (95% CI,.8–2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers, a combined IFR in patients <70 years is estimated at 89 per 100 000 (95% CI, 72–211) infections. Conclusions The IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR is likely severalfold lower than the current estimate. We have initiated real-time nationwide anti–SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic. [ABSTRACT FROM AUTHOR]