question: Healthcare workers (HCWs) stand at the frontline for fighting coronavirus disease 2019 (COVID-19) pandemic. This puts them at higher risk of acquiring the infection than other individuals in the community. Defining immunity status among health care workers is therefore of interest since it helps to mitigate the exposure risk. This study was conducted between May 20th and 30th, 2020. Eighty-five hospitals across Kingdom of Saudi Arabia were divided into 2 groups: COVID-19 referral hospitals are those to which RT-PCR-confirmed COVID-19 patients were admitted or referred for management (Case-hospitals). COVID-19 nonaffected hospitals where no COVID-19 patients had been admitted or managed and no HCW outbreak (Control hospitals). Next, seroprevalence of severe acute respiratory syndrome coronavirus 2 among HCWs was evaluated; there were 12,621 HCWs from the 85 hospitals. There were 61 case-hospitals with 9379 (74.3%) observations, and 24 control-hospitals with 3242 (25.7%) observations. The overall positivity rate by the immunoassay was 299 (2.36%) with a significant difference between the case-hospital (2.9%) and the control-group (0.8%) (P value <0.001). There was a wide variation in the positivity rate between regions and/or cities in Saudi Arabia, ranging from 0% to 6.31%. Of the serology positive samples, 100 samples were further tested using the SAS2pp neutralization assay; 92 (92%) samples showed neutralization activity. The seropositivity rate in Kingdom of Saudi Arabia is low and varies across different regions with higher positivity in case-hospitals than control-hospitals. The lack of neutralizing antibodies (NAb) in 8% of the tested samples could mean that assay is a more sensitive assay or that neutralization assay has a lower detection limits; or possibly that some samples had cross-reaction to spike protein of other coronaviruses in the assay, but these were not specific to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).