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During the COVID-19 pandemic, healthcare workers (HCWs) are at a considerable risk of being infected with SARS-CoV-2; among them, HCWs from ophthalmology departments are more prone to develop severe symptoms. In Mexico City, the prevalence of SARS-CoV-2 infection among HCWs is 30%. The present work aims to describe the seroprevalence among HCWs at an Ophthalmological Reference Centre in Mexico City. A self-report questionnaire, RT-PCR test and detection of serum IgG/IgM antibodies against SARS-CoV-2 were performed among HCWs at the Institute of Ophthalmology "Conde de Valenciana". A total of 169 HCWs participated in the study. None of the participants declared severe symptoms, and only 15% showed three or more symptoms. The results showed that 32% of the participants were RT-PCR+ (54/169), and 20% (35/169) presented IgG antibodies against SARS-CoV-2. Thirteen percent of the RT-PCR+ subjects were IgG positive, and 7.6% of the RT-PCR− participants were IgG positive. The presence of three or more symptoms correlated with the presence of IgG antibodies, as well as Ct values of < 32 (p < 0,05). Most of the HCW cohort showed mild symptoms, and 69% of the RT-PCR+ participants did not show IgG antibodies against SARS-CoV-2. Seroprevalence was significantly associated with the presentation of COVID-19-associated symptoms. [ABSTRACT FROM AUTHOR]