Aims: Alterations in mitochondrial DNA (mtDNA) have been implicated in carcinogenesis and tumor progression. We here evaluated the diagnostic and prognostic potential of mtDNA as a biomarker for breast cancer. Methods: Using multiplex real-time polymerase chain reaction, nuclear DNA (nDNA) and mtDNA levels in serum, buffy coat, tumor, and tumor-adjacent tissue samples from 50 breast cancer patients were determined and assessed for associations with clinicopathological features. To evaluate mtDNA as a biomarker for distinguishing between the four sample types, we created receiver operating characteristic (ROC) curves. Results: The mtDNA levels in buffy coat were significantly lower than in other sample types. Relative to tumor-adjacent tissue, reduced levels of mtDNA were identified in buffy coat and tumor tissue but not in serum. According to ROC curve analysis, mtDNA levels could be used to distinguish between buffy coat and tumor-adjacent tissue samples with good sensitivity (77%) and specificity (83%). Moreover, mtDNA levels in serum and tumor tissue were positively associated with cancer TMN stage. Conclusions: The mtDNA levels in blood samples may represent a promising, non-invasive biomarker in breast cancer patients. Additional, large-scale validation studies are required to establish the potential use of mtDNA levels in the early diagnosis and monitoring of breast cancer.