Aim: Identification of mutations and polymorphisms in the cytochrome b gene (Cyb) of mitochondrial DNA (mtDNA) in canine mast cell tumours and determinatiion of their association with the process of neoplastic transformation. Materials and Methods: The samples comprised tumour tissues and blood obtained from 34 dogs of various breeds. Mutations and polymorphisms in the Cyb gene were detected using amplification and sequencing methods. Results: Heteroplasmic mutations were detected at seven positions of mtDNA in 86% of the individuals. Blood and tumour heteroplasmy were recorded at five nucleotide positions of the Cyb gene, whereas tumour heteroplasmy was detected at two positions. Polymorphisms were detected at 14 Cyb gene positions in in the blood of 91% of dogs with mast cell tumours. Conclusion: The presence of numerous mutations and polymorphisms of Cyb in the blood and tumour tissues and the high frequency of heteroplasmy indicate their involvement in the process of neoplastic transformation in dogs.