Study objective: We determine the percentage of diagnosed and undiagnosed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among a sample of US emergency department (ED) health care personnel before July 2020. Methods: This was a cross-sectional analysis of ED health care personnel in 20 geographically diverse university-affiliated EDs from May 13, to July 8, 2020, including case counts of prior laboratory-confirmed coronavirus disease 2019 (COVID-19) diagnoses among all ED health care personnel, and then point-in-time serology (with confirmatory testing) and reverse transcriptase–polymerase chain reaction testing in a sample of volunteers without a previous COVID-19 diagnosis. Health care staff were categorized as clinical (physicians, advanced practice providers, and nurses) and nonclinical (clerks, social workers, and case managers). Previously undiagnosed infection was based on positive SARS-CoV-2 serology or reverse transcriptase–polymerase chain reaction result among health care personnel without prior diagnosis. Results: Diagnosed COVID-19 occurred in 2.8% of health care personnel (193/6,788), and the prevalence was similar for nonclinical and clinical staff (3.8% versus 2.7%; odds ratio 1.5; 95% confidence interval 0.7 to 3.2). Among 1,606 health care personnel without previously diagnosed COVID-19, 29 (1.8%) had evidence of current or past SARS-CoV-2 infection. Most (62%; 18/29) who were seropositive did not think they had been infected, 76% (19/25) recalled COVID-19–compatible symptoms, and 89% (17/19) continued to work while symptomatic. Accounting for both diagnosed and undiagnosed infections, 4.6% (95% confidence interval 2.8% to 7.5%) of ED health care personnel were estimated to have been infected with SARS-CoV-2, with 38% of those infections undiagnosed. Conclusion: In late spring and early summer 2020, the estimated prevalence of severe acute respiratory syndrome coronavirus 2 infection was 4.6%, and greater than one third of infections were undiagnosed. Undiagnosed SARS-CoV-2 infection may pose substantial risk for transmission to other staff and patients.