Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (≤1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.