The aim of the study was to find associations between the process of neoplastic transformation and mtDNA mutations/polymorphisms, i.e. factors with potential prognostic significance, and to determine their impact on the biochemical properties, as well as structural, and functional properties of proteins. Blood and neoplastic tissue samples were collected from a 9-year-old Labrador dog with a diagnosed malignant mammary tumour. Next-generation genome sequencing (NGS) of the entire mitochondrial genome was performed using Illumina technology, and bioinformatics analyses were carried out. This is the first report demonstrating the application of NGS in the analysis of the canine mtDNA genome in neoplastic disease. The proposed strategy is innovative and promising. For the first time in the literature, the sequence of 29 genes was analysed to determine their association with the prevalence of tumour. In total, 32 polymorphic loci and 15 mutations were identified. For the first time, as many as 24 polymorphisms and all the mutations have been described to be associated with the neoplastic process in dogs. Most polymorphisms/mutations were found in the D-loop (31% of the polymorphisms and 93% of the mutations) and the