The aim of the nationwide prospective seroconversion (PROSECO) study was to investigate the dynamics of anti-SARS-CoV-2 IgG antibodies in the Czech population. Here we report on baseline prevalence from that study. The study included the first 30,054 persons who provided a blood sample between October 2020 and March 2021. Seroprevalence was compared between calendar periods, previous RT-PCR results and other factors. The data show a large increase in seropositivity over time, from 28% in October/November 2020 to 43% in December 2020/January 2021 to 51% in February/March 2021. These trends were consistent with government data on cumulative viral antigenic prevalence in the population captured by PCR testing – although the seroprevalence rates established in this study were considerably higher. There were only minor differences in seropositivity between sexes, age groups and BMI categories, and results were similar between test providing laboratories. Seropositivity was substantially higher among persons with history of symptoms (76% vs. 34%). At least one third of all seropositive participants had no history of symptoms, and 28% of participants with antibodies against SARS-CoV-2 never underwent PCR testing. Our data confirm the rapidly increasing prevalence in the Czech population during the rising pandemic wave prior to the beginning of vaccination. The difference between our results on seroprevalence and PCR testing suggests that antibody response provides a better marker of past infection than the routine testing program.