JC and BK polyomaviruses (JCV and BKV) infect humans and can cause severe illnesses in immunocompromised patients. Merkel cell polyomavirus (MCPyV) can be found in skin carcinomas. In this study, we assessed the occurrence of serum antibodies against MCPyV, BKV, and JCV polyomaviruses in a healthy population of the Czech Republic. Serum samples from 991 healthy individuals (age range: 6-64 years) were examined by enzyme-linked immunoassay (ELISA) using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against MCPyV, JCV, and BKV were found in 63%, 57%, and 69%, respectively, of this population. For all three viruses, these rates were associated with age; the occurrence of antibodies against MCPyV and JCV was highest for those older than 59 years, while the occurrence of antibodies against BKV was highest in those aged 10-19 years and 20-29 years. This is the first large study to determine the seroprevalence rates for BKV, JCV, and MCPyV polyomaviruses in the general Czech Republic population.