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Background: Previous studies indicate hypocalcaemia as a potential diagnostic and prognostic marker of corona-virus disease 2019 (COVID-19). Our aim was to investigate these relations in more detail in a large test cohort and an independent validation cohort., Methods: We retrospectively included 2792 COVID-19 suspected patients that presented to the emergency department (ED) of two hospitals. Plasma calcium and ionized plasma calcium levels were compared between COVID-19 positive and negative patients, and between severe and non-severe COVID-19 patients using univariate and multivariate analyses in the first hospital ( N  = 1363). Severe COVID-19 was defined as intensive care unit (ICU) admission or death within 28 d after admission. The results were validated by repeating the same analyses in the second hospital ( N  = 1429)., Results: A total of 693 (24.8%) of the enrolled patients were COVID-19 positive, of whom 238 (34.3%) had severe COVID-19. In both hospitals, COVID-19 positive patients had lower plasma calcium levels than COVID-19 negative patients, regardless of correction for albumin, in univariate and multivariate analysis (Δ0.06-0.13 mmol/L, p  < .001). Ionized plasma calcium concentrations, with and without correction for pH, were also lower in COVID-19 positive patients in multivariate analyses (Δ0.02-0.05 mmol/L, N  = 567, p  < .001). However, we did not find a significant association between COVID-19 disease severity and plasma calcium in multivariate analyses., Conclusions: Plasma calcium concentrations were lower in COVID-19 positive than COVID-19 negative patients but we found no association with disease severity in multivariate analyses. Further understanding of plasma calcium perturbation may facilitate the development of new preventive and therapeutic modalities for the current pandemic.