The necessity of post-transplant monitoring for donor-specific antibodies (DSAs) is unclear. This study evaluates the clinical relevance of post-transplantation donor-specific HLA antibodies in pediatric renal transplant recipients, aiming at better stratification of patients at risk of graft dysfunction and better recommendations for post-transplant monitoring. A cohort of 68 pediatric kidney recipients, involving 76 transplantations between 2004 and 2014, was studied retrospectively. All patients were screened for HLA antibodies at 1, 3, 6, and 12 months after transplantation and yearly thereafter. Samples testing positive were further analyzed to detect DSA. A biopsy was performed on clinical indication. We studied the baseline characteristics of the patients with biopsy, with DSA, and with rejection. We assessed the effect of post-transplant DSA on clinical outcome, including antibody-mediated acute rejection and GFR decrease. In our cohort, the prevalence of DSA was 19% (13/68 transplantations). Most patients with HLA antibodies after transplantation were DSA-positive (76%; 13/17). A clear association between DSA and subsequent rejection was found. At the end of the study period, a significantly lower GFR was found in patients with biopsy, DSA, or rejection. Based on our observations, we recommend routine post-transplantation screening for HLA and DSA. The presence of DSA justifies a renal biopsy even in the absence of clinical signs of rejection.