The impact of subclinical viral infection on chronic allograft injury in the pediatric renal transplant population is not well defined. We prospectively assessed cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia by monthly PCR in 55 pediatric renal transplant recipients for the first 2 years after transplantation. Subclinical CMV and EBV infection occurred in 22 and 36%, respectively. Multivariable linear regression analysis suggested that both subclinical CMV and EBV infection independently associate with significant declines in GFR during the first 2 years after transplantation. CMV seronegativity associated with a significantly greater decline in GFR than seropositivity (P < 0.01). Subclinical CMV infection and subclinical EBV infection each associated with approximately fourfold greater odds of histologic evidence of chronic allograft injury (odds ratio 4.61 [95% confidence interval 1.18 to 18.07] and odds ratio 4.33 [95% confidence interval 1.34 to 14.00], respectively). An increase in viral load of CMV or EBV also associated with increased risk for moderate to severe chronic allograft injury. Taken together, these results demonstrate an association between subclinical CMV and EBV infections, which occur despite standard antiviral prophylaxis, and chronic allograft injury in pediatric renal transplant recipients.