Polyomavirus JC (JCPyV) is largely excreted by the human population through the urinary route and has been recognized as a potential viral marker for human waste contamination. This study aims to investigate the dissemination of JCPyV in waste water from a sewage treatment plant (STP) located in Rio de Janeiro, Brazil, and to describe the prevalence of JCPyV subtypes currently present in this population. Raw and treated sewage samples were collected bimonthly during one year, and examined for the presence of JCPyV using nested polymerase chain reaction (nPCR) and quantitative real time PCR (qPCR). JCPyV was detected by nPCR in 96% and 43% of raw and treated sewage samples, respectively. The concentration of JCPyV present in the samples ranged from 1.2 × 103 to 3.2 × 105 and 2.6 × 102 to 6.2 × 103 genome copies per 2 ml of concentrated raw and treated sewage sample, respectively. The strains were characterized and the obtained nucleotide sequences indicated that the detected JCPyV strains clustered with subtypes of East African, West African and European origin. To our knowledge, this is the first study describing the incidence and diversity of JCPyV strains in raw and treated sewage in Brazil.