Limited prospective SARS-CoV-2 data in children regarding the impact of Omicron variant in seropositivity have been reported. We investigated SARS-CoV-2 seropositivity in children between 1 September 2021 and 30 April 2022, representing Delta and Omicron predominance periods. Serum samples from children admitted to the major tertiary Greek pediatric hospital for any cause, except for COVID-19, were randomly collected and tested for SARS-CoV-2 natural infection antibodies against nucleocapsid antigen (Elecsys® Anti-SARS-CoV-2 reagent). A total of 506/1312 (38.6%) seropositive children (0-16 years) were detected [males: 261/506(51.6%); median age (IQR): 95.2 months(24-144)]. Seropositivity rates (%) increased from Delta to Omicron period from 29.7% to 48.5% (P-value<0.0001). Seropositivity increased for all age groups, except for the age group of 0-1 year (P-value:0.914). The highest seropositivity rate was detected in April 2022 (52.6%) and reached 73.9% specifically for the age group 12-16 years. No significant differences were detected in seropositivity with respect to gender, origin, or hospitalization status. Median (IQR) antibody titers were higher in the Omicron vs Delta period in all age groups, especially in 12-16 years [32.2 COI (7-77.1) vs 11.4 COI(2.8-50.2), Pvalue:0.009]. During Omicron variant period increased SARS-CoV-2 seropositivity was detected in pediatric population, especially in adolescents, implicating either increased transmissibility or reinfection rates.