Previously, we found that mitochondrial DNA (mtDNA) is elevated in adults with chronic graft-versus-host disease (cGvHD), acting as an endogenous source of TLR9 agonists to augment B cell responses. To validate this in children, we evaluated mtDNA plasma expression in a large pediatric cohort (ABLE /PBMTC 1202 study). Plasma cell-free mtDNA (cf-mtDNA) copy numbers were measured in 202 pediatric patients by quantitative Droplet Digital polymerase chain reaction (ddPCR). Two evaluations were performed: a) before the onset of cGvHD or late aGvHD at day 100 +/- 14 days and b) at the time of onset cGvHD compared to time-matched non-cGvHD controls. We found that cf-mtDNA copy numbers were not affected by immune reconstitution post-HSCT, but were higher on day 100 before the onset of late aGvHD and at the onset of cGvHD. We found cf-mtDNA was not impacted by previous aGvHD, but correlated with the early onset, NIH moderate/severe cGvHD, and did not correlate with other immune cell populations, cytokines, or chemokines, but did with the metabolites spermine and taurine. Children, like adults, have elevated plasma cf-mtDNA concentrations at the early onset of cGvHD, especially in NIH moderate/severe cGvHD, elevation with late aGvHD, and associates with metabolites involved in mitochondrial function.