Polyomavirus (PyV) infection usually persists without any symptoms in normal individuals. In immunocompromised patients including patients with systemic lupus erythematosus (SLE), PyV reactivation occurs with a high prevalence and can cause severe clinical complications. In this study, reactivation of six PyV [JC, BK, WU, KI, merkel cell (MC) and trichodysplasia spinulosa (TS)] was investigated in terms of prevalence, clinical implications and correlation with urine transforming growth factor (TGF)-β1 expression in 50 SLE patients aged less than 18 years. Clinical characteristics were obtained from medical record review. PyV viruria was assessed by nested polymerase chain reaction. Urine TGF-β1 was measured with ELISA. The mean age was 13 ± 2.8 years. The prevalence of JC and BK viruria was 16% and 32%, respectively. WU, KI, MC and TS were not isolated from any urine specimens. Co-reactivation of 2 PyV was not detected. Urine TGF-β1 levels in patients with JC viruria, with BK viruria and without PyV viruria were 0.27 ± 0.09, 0.10 ± 0.05 and 0.13 ± 0.09 ng/mg of urine creatinine, respectively. Cumulative doses of cyclophosphamide per body weight and urine TGF-β1 levels were higher in JC viruria than in other groups (p < 0.05). The prevalence of JC and BK reactivation was higher in pediatric patients with SLE than in the normal population. JC reactivation in pediatric patients with SLE was correlated with the administration of high-dose cyclophosphamide and increased urine TGF-β1 levels. Surveillance of JC reactivation is recommended in pediatric patients with chronic and severe SLE receiving high-dose cyclophosphamide.