The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of glioblastoma (GB) patients are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GB (n=64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment naïve GB PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2x coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with Temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% versus 24%) and allowed detection in cerebrospinal fluid (CSF) and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GB where current methods have largely failed.