<jats:title>Abstract</jats:title><jats:p>Recent studies indicate that urinary mitochondrial DNA (mtDNA) is predictive of ischemic AKI and is related to delayed graft function (DGF) in renal transplantation. Nevertheless, the clinical implications and prognostic value of urinary mtDNA in kidney transplantation remain undetermined. Here, we aimed to evaluate the associations between cell-free mtDNA and clinical parameters, including pathological findings in allograft biopsy and post-transplant renal function. A total of 85 renal transplant recipients were enrolled, and blood and urine samples were collected at a median of 17 days after transplantation. Cell-free nuclear and mtDNA levels were measured by quantitative polymerase chain reaction for <jats:italic>LPL</jats:italic> and <jats:italic>ND1</jats:italic> genes. Urinary cell-free mtDNA levels were significantly higher in patients with DGF (<jats:italic>P</jats:italic> < 0.001) and cases of deceased donor transplantation (<jats:italic>P</jats:italic> < 0.001). The subjects with acute rejection showed higher urinary mtDNA levels than those without abnormalities (<jats:italic>P</jats:italic> = 0.043). In addition, allograft functions at 9- and 12-month post-transplantation were significantly different between tertile groups of mtDNA independent of the presence of DGF or acute rejection, showing significantly better graft outcome in the lowest tertile group. Urinary cell-free mtDNA levels during the early post-transplant period are significantly associated with DGF, acute rejection in graft biopsy, and short-term post-transplant renal function.</jats:p>