Background: The BK Polyomavirus (BKPyV) and JC polyomavirus (JCPyV) infections are widespread in human population and have been associated with severe kidney and brain disorders, respectively. The viruses remain latent primarily in reno-urinary tract, reactivating only in case of a compromised immune system. The seroepidemiology and molecular prevalence of BKPyV and JCPyV have been widely studied both in healthy and immunocompromised patients worldwide. However, data regarding the prevalence of these viruses in the immunocompetent or apparently healthy Pakistani population is lacking. Herein, we present the first ever report on quantitative prevalence of BKPyV and JCPyV in the peripheral blood of a randomly selected cohort of healthy Pakistani population. Methods: A total of 266 whole blood samples were examined. The subjects were divided into three age groups: ≤ 25 years (young), 26-50 years (middle) and ≥ 51 years (elder). Absolute real time PCR assay was designed to quantify the BKPyV and JCPyV viral copy numbers in the range of 106 to 100 copies/mL. Results: Overall, BKPyV was detected in 27.1% (72/266) individuals while JCPyV in 11.6% (31/266) indicating significant difference (p < 0.005) in the distribution of these two viruses. The prevalence of BKPyV significantly decreased from 51% (49/96) in young age group to 8.2% (7/85) in eldest age group. Whereas, JCPyV positivity rate slightly increased from 8.3% (8/96) in young age group to 11.8% (10/85) in elder age group. The median viral load was calculated as 6.2 log and 3.38 log copies/mL of blood for BKPyV and JCPyV, respectively. Notably, no significant difference in viral load of either of the subtypes was found between different age groups. Conclusion: The current study provides an important baseline data on the prevalence and viral load of circulating BKPyV and JCPyV in Pakistani population. The prevalence and viral load of BKPyV was comparatively higher than JCPyV. The prevalence of BKPyV significantly decreased with increase in age while JCPyV positivity rate slightly increased with increasing age. Viral load of both BKPyV and JCPyV was not correlated with the individual ages.