Cell-free DNA (cfDNA) is a marker of organ injury and immune response. DNA methylation is an epigenetic regulator of gene expression. Here, we elucidate total plasma cfDNA methylation from kidney transplant recipients in presence versus absence of rejection. In doing so, we exploit cfDNA as a real-time biomarker to define molecular pathways of rejection. Twenty plasma cfDNA samples from pediatric kidney transplant recipients were collected at allograft biopsy. Differentially methylated cytosine residues (>
20 % methylation difference, q-value <
0.05) were identified in presence (N = 7) versus absence (N = 9) of acute rejection. Separate analyses were performed comparing borderline rejection (N = 4) to rejection and non-rejection. In rejection versus non-rejection, there were 1269 differentially methylated genes corresponding to 533 pathways. These numbers were 4-13× greater than comparisons against borderline samples. Enriched pathways between rejection and non-rejection samples were related to immune cell/inflammatory response, lipid metabolism, and tryptophan-kynurenine metabolism, suggesting differential methylation of these pathways contributes to rejection.