Abstract The aetiology and pathogenesis of many canine tumours are likely to be similar to cancers found in humans. This study aimed to evaluate a plausible link between changes in the tRNA-Leu (UUR) gene and the carcinogenesis process in dogs with mammary gland tumours. The whole mitochondrial DNA (mtDNA) isolated from blood and tumour tissues of 13 dogs with malignant mammary gland tumours was sequenced. The present work is the first report showing that some polymorphisms might occur at the corresponding positions in the human and canine mtDNA genome, which in turn may provoke similar deleterious effects. The homology between the human MT-TL1 and canine tRNA-Leu (UUR) genes was 84%. After resequencing of the whole mitochondrial DNA genome with the use of the NGS technology, two polymorphisms in two haplotypes were identified: m.2683G>A (observed in 18 out of 27 samples) and m.2678_2679insG (27 out of 27 samples). The m.2683G>A polymorphism corresponded to a deleterious change at m.3243A>G, which is linked with MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, Stroke-like episodes) syndrome and with different types of cancers in humans as well. The comparative analysis of MT-TL1 and tRNA-Leu (UUR) led us to hypothesise that the m.2678_2679insG and m.2683G>A polymorphisms might influence the dog’s condition and might be linked with tumourigenesis, as observed in humans.