Recent studies have demonstrated a significant role of mitochondrial DNA (mtDNA) defects in the pathogenesis of many human and some canine tumours. The aim of this study was to identify mutations in the ND2 and ND4 mitochondrial genes in canine mast cell tumours and determine their association with the process of neoplastic transformation and the phenotypic traits of dogs. In total, 136 gene sequences from 68 biological samples, including blood and neoplastic tissue samples from 34 dogs with diagnosed MCTs, were analysed. The study consisted in DNA sequencing of the ND2 and ND4 genes as well as bioinformatics and statistical analyses. For the first time, muta-tions in NADH-dehydrogenase genes were detected in dogs with MCTs. In total, 22 polymorphic loci and 19 mutations in the ND2 and ND4 genes were identified. The majority of the identified mutations were homoplasmic, and tumour heteroplasmy was detected in eight nucleotide positions in three dogs. Seven of the ND2 mutations and two of the ND4 mutations caused an amino acid change. The changes in non-synonymous protein-coding SNPs did not exert an adverse effect on proteins. A statistically significant correlation of the presence of mutations/polymorphisms with the sex, age, and size of the dogs and the tumour location was demonstrated. Polymorphisms and mutations in NADH-dehydrogenase genes, including mastocyte-specific changes, in canine mast cell tumours that had not been reported earlier in the literature were identified. Some of these changes may imply that these are the hotspot mutations in canine mast cell tumours. It can-not be excluded that the molecular changes are directly associated with the development of mast cell tumours, and further investigations are needed to verify whether they can become molecular markers of MCTs in the future.