Understanding mitochondrial biology is a fundamental research goal in human genetics and medicine. The use of mitochondria to serve as a biomarker is rapidly expanding in disciplines ranging from cancer, rare metabolic diseases, aging, the tracing of human migration patterns in antiquity, population characterization using maternal markers, and human identification. Mitochondrial DNA (mtDNA) mutations occur frequently in cancer, and there is an important need for validating mtDNA mutations as cancer biomarkers for the detection of early-stage disease. Although a few studies have suggested tissue-specific mtDNA mutations, there is no single mutational hotspot associated with the wide spectrum of cancer patients; hence, sequencing the entire mitochondrial genome and further characterization of the multiple deletions associated with tumors is required to detect the mutation load on an individual basis. Microarray-based technology provides a reliable and rapid method to detect all mutations of the entire mitochondrial genome. In addition to microarray-based sequencing, real-time PCR is an important method for deletion analysis. Mutations throughout the mitochondrial genome are recurrent events in primary tumor tissues and in corresponding non-invasively collected body fluids. Thus, mtDNA mutation analysis may provide a molecular tool for the early detection and prognosis of cancer. Recent findings have verified that relatively simple diagnostic tests for detecting mtDNA mutations, involving mitochondrial microarray chips and/or real-time PCR bioassays, have exciting predictive potential for cancer detection and prognosis.