The aim of this study was to determine molecular defects in mitochondrial DNA(mtDNA) with the use of large-scale genome analysis in malignant canine mammarygland tumours and indicate whether these changes were linked with the carcinogenesis process. With the use of the NGS technology, we sequenced 27 samples of mtDNA isolated from blood and tumours obtained from 13 dogs with mammary gland tumours. The total number of mutations and polymorphisms in the analysed mitochondrialgenomes was 557. We identified 383 single nucleotide polymorphisms (SNP), 32 indels (or length polymorphisms), 4 mutations, 137 heteroplasmic positions and 1 indel muta-tion. The highest variability (132 changes) was observed in the variable number oftandem repeats (VNTR) region. The heteroplasmy rate in VNTR varied among individ-uals and even between two tumours in one organism. Our previous study resulted indetermination of a probable CpG island in this region, thus it is not excluded that thesechanges might alter mtDNA methylation. Only theATP8gene was not affected by anypolymorphisms or mutations, whereas theCOX1gene had the highest number of polymorphisms from all protein-coding genes. One change m.13594G>A was detected in aregion spanning two genes:ND5andND6, from which a deleterious effect was observed for the ND5 protein. Molecular changes were frequently observed in themTΨC loop, which is thought to interact with ribosomal RNA