Human mitochondrial DNA (mtDNA) encodes 13 polypeptides essential for oxidative phosphorylation. Because of the unique features of "replicative segregation" and "threshold expression" of mtDNA genetics, identification of homoplasmy versus heteroplasmy status is critical. Results from various detection methods may lead to different interpretations on formation or outcome of mtDNA mutations, such as the conclusion of somatic mutation versus genetic drift in cancers. Denaturing high-performance liquid chromatography (DHPLC) and pyrosequencing (PSQ) have recently been employed to confirm the presence of heteroplasmy of mtDNA because of their high sensitivity in detecting heteroplasmic mutations compared with direct sequencing. Moreover, PSQ has superior ability in quantifying percentage of heteroplasmy. However, there could be disagreement between these two techniques and several issues specific for mtDNA should be taken into consideration. Particularly, DHPLC analysis should be more prone to be interfered by nuclear mitochondrial sequences (Numts), if it is coamplified with mtDNA, than PSQ analysis.