Objectives: The objective of this study was to perform a large seroprevalence SERO survey on severe acute respiratory syndrome MESHD coronavirus 2 (SARS-CoV-2) among Danish healthcare workers to identify high risk groups. Design: Cross-sectional survey. Setting: All healthcare workers and administrative personnel at the seven hospitals, pre-hospital services and specialist practitioner clinics in the Central Denmark Region were invited by e-mail to be tested for antibodies SERO against SARS-CoV-2 by a commercial SARS-CoV-2 total antibody SERO enzyme-linked immunosorbent assay SERO ( ELISA SERO, Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing, China). Participants: A total of 25,950 participants were invited. Of these, 17,987 (69%) showed up for blood SERO sampling, and 17,971 had samples available for SARS-CoV-2 antibody SERO testing. Main outcome measures: 1) Prevalence SERO of SARS-CoV-2 antibodies SERO; 2) Risk factors for seropositivity; 3) Association of SARS-CoV-2 RNA and antibodies SERO. Results: After adjustment for assay sensitivity SERO and specificity, the overall seroprevalence SERO was 3.4% (CI: 2.5%-3.8%). The seroprevalence SERO was higher in the western part of the region than in the eastern part (11.9% vs 1.2%, difference: 10.7 percentage points, CI: 9.5-12.2). In the high prevalence SERO area, the emergency MESHD departments had the highest seroprevalence SERO (29.7%) while departments without patients or with limited patient contact had the lowest seroprevalence SERO (2.2%). Multivariable logistic regression analysis with age TRANS, sex, and profession as the predictors showed that nursing staff, medical doctors, and biomedical laboratory scientists had a higher risk than medical secretaries, who served as reference (OR = 7.3, CI: 3.5-14.9; OR = 4., CI: 1.8-8.9; and OR = 5.0, CI: 2.1-11.6, respectively). Among the total 668 seropositive participants, 433 (64.8%) had previously been tested for SARS-CoV-2 RNA, and 50.0% had a positive RT-PCR result. A total of 98% of individuals who had a previous positive viral RNA test were also found to be seropositive. Conclusions: We found large differences in the prevalence SERO of SARS-CoV-2 antibodies SERO in staff working in the healthcare sector within a small geographical area of Denmark and signs of in-hospital transmission TRANS. Half of all seropositive staff had been tested positive by PCR prior to this survey. This study raises awareness of precautions which should be taken to avoid in-hospital transmission TRANS. Additionally, regular testing of healthcare workers for SARS-CoV-2 should be considered to identify areas with increased transmission TRANS. Trial registration: The study is approved by the Danish Data Protection Agency (1-16-02-207-20).