Formalin-fixed paraffin embedded (FFPE) tissue is the most common histopathological technique for tumour assessment in the veterinary pathology. Due to easy availability of FFPE blocks, the aim of this retrospective study was to identify molecular defects caused by mutations in mitochondrial DNA (mtDNA) in cases of canine malignant mammary gland tumours (MGTs). DNA was isolated from 88 FFPE samples, then quantity and quality analyses were performed. Amplification was conducted on 10 selected fragments of 8 mitochondrial genes (COX1, COX3, ND1, ND2, ND4, ND4L, ND5 and ATP6), and finally obtained PCR products were sequenced. After that, bioinformatic analyses of nucleotide and protein sequences were done. The total number of observed mtDNA changes was 512 based on 23 changed nucleotide positions including 20 substitutions, 1 indel mutation and 4 heteroplasmic sites. The most commonly occurred mtDNA variants were m.8807G>A (COX3) and m.9911_9912insGT (ND4L) followed by m.3196T>C (ND1), m.5444T>C (COX1) and m.3254G/A (ND1). The changes in the protein-coding genes were mostly synonymous, and nonsynonymous changes were mostly tolerant and did not lead to alterations in protein properties. The number of mtDNA changes per sample varies from 2 to 13, and not depended on histopathological tumour type, tumour grading, age or breed of dog.