The aim of this study was to identify mutations in the D-loop region of mtDNA in head, neck, and limb tumours in dogs, and determination of their relationship with the process of neoplastic transformation. Blood and tumour tissue samples from 19 dogs with diagnosed sporadic malignant tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA D-loop, and bioinformatic analyses were performed. Five mutations and 19 polymorphisms were observed in 68.42% of all tumours. Polymorphic variants were noted in 42.86% of the head and neck tumours and in 58.33% of the limb tumours. Mutations were observed in 21.05% of dogs. The mutations were found in 28.57% of the head and neck tumours and in 16.66% of the limb tumours. The mutations were identified in 50% of the studied epithelial cancers. In the mesenchymal tumours, no mutations in the D-loop region were observed. Mitochondrial haplotype A17 was found in over 40% cases of limb tumours. No association between the age, breed, sex, type of tumour, and detected polymorphic variants were observed. Different mutational changes in the D-loop sequences of mtDNA identified in the blood and tumour tissues may indicate a relationship between the type of tumour and individual changes in the D-loop nucleotide sequences of mtDNA.