Mitochondrial DNA (mtDNA) alterations were reported in many cancers but their roles in oncogenesis are still debated. We aimed to examine qualitative and quantitative mtDNA modifications and oxidative stress in normal and cancer cells.23 leukemia patients and 18 healthy subjects were recruited from the hospital of Azziza Othmana (Tunisia). Mitochondrial D-loop was sequenced, mtDNA level was determined by Q-PCR, the oxidative stress was assessed by a fluorescent probe and the mitochondrial transcription factor A (mTFA) level was quantified.No somatic mutation was evidenced in leukemia cells compared to non-malignant cells. However, a significant higher level of mtDNA associated with an increase of mTFA expression and reactive oxygen species (ROS) production were measured in patients' cells compared to non-malignant cells.In conclusion, our results don't support the role of mtDNA mutations in leukemogenesis but increase of mtDNA and ROS levels would be molecular signatures of leukemia.