Abstract: Methods, compositions, and kits for detecting mutations in the entire human mitochondrial genome. A preferred method includes amplifying mtDNA from a biological sample by polymerase chain reaction of total DNA using a plurality of pre-selected primer pairs to generate overlapping amplicons; cleaving the amplicons using restriction enzymes to produce fragments suitable for analysis by denaturing high performance liquid chromatography (DHPLC); denaturing and re-annealing the amplicons; and fragment analysis by DHPLC to detect the presence or absence of heteroduplex molecules.