Background: Childhood AML is a cytogenetically and morphologically heterogeneous disease. Mitochondrial DNA (mtDNA) has a 10- to 20-fold greater susceptibility to genetic mutation. We speculated that aberrations of mtDNA in total bone marrow cell would be associated with risk of developing childhood AML. Patients and Methods: To detect alteration of mtDNA sequence, sequencing and gene scan analyses were performed in 55 patients with initially diagnosed childhood AML and 55 matched controls. Direct sequencing of mtDNA control region, CYTB, and tRNA leucine 1 genes were performed to determine the mtDNA sequence alteration between AML cells and non-AML cells in the same patients. mtDNA copy number was measured using real time PCR and the level of intracellular ROS was measured. MitoTracker Green and Red probe were used. Results: Numerous sequence alterations of mtDNA were only found in patient’s cells. Instability of mtDNA minisatellites mainly occurred in the 303 poly C and 16184 poly C minisatellites. mtDNA copy number was significantly elevated in patients (p0.0001). The level of intracellular ROS, mitochondrial mass and its membrane potential were increased in patients. The frequency of mtDNA 4977bp large deletion was significantly increased in patients (p0.01). There was a dose-response relationship between tertiles of mtDNA copy number and risk of developing childhood AML (OR, 95%: 1.0; 1.28; and 7.67, respectively; p=0.001). Patients harboring large amount of mtDNA 4977bp large deletion showed shorter event-survival rate (p=0.0585) than those with small amount. Conclusion: mitochondrial aberrations were markedly increased in childhood AML patients compared with normal controls. Polymorphisms at and around the areas of transcriptional control may potentially contribute to tumorigenesis. Moreover, amount of 4977bp large deletion might contribute to the poor prognosis of childhood AML.