The occurrence of heteroplasmy and mixtures is technically challenging for the analysis of mitochondrial DNA. More than that, observed mutations need to be carefully interpreted in the light of the phylogeny as mitochondrial DNA is a uniparental marker reflecting human evolution. Earlier attempts to explain the role of mtDNA in cancerous tissues led to substantial confusion in medical genetics mainly due to the presentation of low sequence data quality and misinterpretation of mutations representing a particular haplogroup background rather than being cancer-specific. The focus of this study is to characterize the extent and level of mutations in breast cancer samples obtained by tissue microdissection by application of an evaluated full mtDNA genome sequencing protocol. We amplified and sequenced the complete mitochondrial genomes of microdissected breast cancer cells of 15 patients and compared the results to those obtained from paired non-cancerous breast tissue derived from the same patients. We observed differences in the heteroplasmic states of substitutions between cancerous and normal cells, one of which was affecting a position that has been previously reported in lung cancer and another one that has been identified in 16 epithelial ovarian tumors, possibly indicating functional relevance. In the coding region, we found full transitions in two cancerous mitochondrial genomes and 12 heteroplasmic substitutions as compared to the non-cancerous breast cells. We identified somatic mutations over the entire mtDNA of human breast cancer cells potentially impairing the mitochondrial OXPHOS system.