Abstract: The present disclosure provides methods for detecting macro-heteroplasmy and/or micro-heteroplasmy in mitochondrial DNA. The methods can include detecting or monitoring the presence of heteroplasmy, and/or identifying a threshold level of heteroplasmy. In addition, the methods can be used for diagnosing a mitochondrial related disease or disorder, as well as for monitoring the efficacy of a therapy affecting mitochondrial DNA (mtDNA) in a subject having or suspected of having heteroplasmy.