he aim of this study was to identify molecular defects caused by mutations in mitochondrial dna in cases of recurrent and multiple canine tumours. We presented molecular differences in the mtDNA genome for two tumours observed in different body parts of five dogs and throughout time in the case of the recurrence. Mitochondrial dna was sequenced on an illumina Miseq sequencer using a 600-cycle kit in a paired-end mode targeting at least 100x coverage. the sequences obtained were subjected to bioinformatic analyses in order to determine mutation and polymorphic sites within the analysed mtdna genome in the tumour tissue. the total amount of changes: single nucleotide polymorphisms (snps), indels, mutations, and heteroplasmy detected in this study was 329. ten polymorphisms were found in all analysed samples: ins.2679_2680g (tRNA-Leu (UUR)), m.5367c>t (COX1), m.5444t>c (COX1), m.6065a>g (COX1), m.8368c>t (ATP6), m.8807g>a (COX3), ins.9913_9914tg (ND4L), m.13299t>a (ND5), m.15814c>t, and m.16418a>g (control region). interest- ingly, the highest number of differences in the mtdna genome was observed between non-cancerous pyogranuloma tissue and epithe- lioma glandulae sebacei. the mutations in the non-cancerous tissue were mainly found in positions where polymorphisms were observed in blood and tumour tissue. the lowest number of changes was observed for the youngest analysed dog, which may indicate that some changes appeared in the mitogenomes with age. there were fewer heteroplasmic alterations in the larger than smaller tumour, which may suggest that the tumour growth is enhanced by genomic instability. the changes in the protein-coding genes were mostly synonymous, and nonsynonymous changes did not lead to alterations in protein properties. new mutations were observed in the post-recurrence tu- mours in comparison with the pre-recurrent tissue and blood.