JC Polyomavirus (JCPyV) has the potential to be used as a viral marker for human waste contamination because at least 40% of the human population excretes this virus through its urine. In addition, each of 6 known subtypes of JCPyV is associated with a specific human ethnicity group, which has allowed for tracing of human migration. This study aims to explore the possibility of linking the genetic diversity of JCPyV with source of human waste. Primary sewage effluent from Irvine Ranch Water District (IRWD) sewage treatment facility was collected and examined for the presence of JCPyV using polymerase chain reaction (PCR). JCPyV was detected by nested PCR using primers specific to the conservative regulatory region in 100% of the 13 samples collected monthly over a year. Only 5 samples were amplifiable by the primers specific to the diverse intergenic region of the virus. Sequence analyses of cloned amplicons from the intergenic region indicated that JCPyV matched subtypes of European, Asian, African and African-American origins. A genotype that is unique from previously identified clinical sample is also revealed. This research suggests the diversity of JCPyV subtypes can be used as a tool to trace the source of human waste contamination.