Somatic mutations in mitochondrial DNA (mtDNA) have been identified in various tumors, including breast cancer. However, their clinicopathological impact on breast cancer still remains unclear. In this study, we re-sequenced the entire mtDNA from breast cancer samples together with paired non-tumorous breast tissues from 58 Taiwanese patients. We identified 19 somatic mutations in the mtDNA coding region of 16 breast cancers. Out of these mutations, 12 of the 19 mutations (63%) are missense or frame-shift mutations that have the potential to cause mitochondrial dysfunction. In combination with our previously study on the D-loop region of mtDNA, we found that 47% (27/58) of the breast cancers harbored somatic mtDNA mutations. Among a total of 40 somatic mutations, 53% (21/40) were located in the D-loop region of the mtDNA, 5% (2/40) were in the ribosomal RNA genes, 5% (2/40) were in the tRNA genes, and 38% (15/40) occurred in mRNA genes. The occurrence of these somatic mtDNA mutations is associated with an older onset age (≥ 50-year old, P = 0.039), a higher TNM stage (P = 0.027), and a higher histological grade (P = 0.012). Multiple logistic regression analysis revealed that an older onset age (P = 0.029) and a higher histological grade (P = 0.006) are significantly correlated with patients having somatic mutations in the mtDNA in their breast cancer sample. In conclusion, our results suggest that somatic mtDNA mutations may play a critical role in the progression of breast cancer.