The spontaneous or mutagen-induced transfer of mitochondrial DNA (mtDNA) into the nuclear genome, as well as changes in mtDNA copy number and intercellular mitochondrial transfer, are in part associated with the development of pathological conditions, including cancer. Therefore, the availability of rapid and easy-to-use tools for identifying mitochondrial exchange and mtDNA integration into the nuclear genome is important. Surprisingly, we were unable to find any commercially available fluorescence in situ hybridization (FISH) probes for mtDNA. Here, we present a detailed protocol for the identification of mtDNA using FISH. FISH analysis of mtDNA enables the identification of mtDNA fragments in human chromosomes and cellular changes in mtDNA copy number that may be important from a diagnostic, clinical, and research perspective.