Background: JC polyomavirus (JCV) is an epitheliotropic and neurotropic virus that identified in relationship with some devastating complications such as progressive multifocal leukoencephalopathy (PML) and linked to colorectal cancer. The aim of current study was to identify the prevalence of JCV in colorectal cancer for the first time in Iran. Methods: This retrospective case-control study was conducted by the hospitals affiliated to Iran University of Medical Sciences, Tehran, Iran from 2011 to 2016. Formalin-fixed paraffin-embedded (FFPE) blocks were used for DNA extraction by QIAamp® DNA FFPE Tissue Kit. The SYBER Green Real-time PCR assay performed by specific primers for JCV T-Large Ag. Melting curve analysis used for evaluation of amplification specificity. Positive control cloned in pTZ57R/T plasmid by Generay Biotechnology system. Results: Of 157 specimens 66 were colorectal cancer by the mean age (y) ± std. deviation 59.35±14.48 and 91 healthy control by the mean age (y) ± std. deviation 57.21±14.66. All 157 specimens tested for JCV T-Large Ag gene by Real-time PCR method and we found that there was not any positive result although the melting analysis showed specificity of positive control amplification. Conclusion: Low prevalence of JCV infection in Iranian CRC population confirmed by the current study results; there was not any JCV positive result in CRC and healthy control groups. Further studies by broader and different populations are recommended