Canine mammary tumours (CMTs) are one of the most frequently observed malignancies in female dogs. Many studies have investigated epigenetic changes in CMT at the nuclear level, yet the role of mtDNA methylation, particularly in the D-loop region, remains largely unexplored. Given the significant importance of the D-loop region in mtDNA replication and transcription, and the previous identification of CpG enrichment there, this preliminary study aimed to assess apparent methylation signals in the mitochondrial D-loop region. We examined CpG, CHG and CHH methylation contexts in matched tumour and normal tissues from six German Shepherd dogs with histologically classified mammary carcinomas using enzymatic methyl-sequencing and high-throughput amplicon sequencing. Overall, CpG and CHH contexts exhibited greater variability between tumour and normal tissues than CHG, which remained relatively stable. Correlation analyses revealed a putative positive relationship between differences in CpG and CHH methylation (Spearman's r = 0.829, p = 0.042). This might suggest a coordinated epigenetic modulation of these two methylation patterns in the carcinogenesis process, yet due to the limited study group (n = 6) the hypothesis should be verified on a larger cohort. These findings demonstrate a plausible existence of intra-individual epigenetic divergence at the mitochondrial level. To our knowledge, this is the first study to assess the influence of mtDNA methylation on CMT, and it supports further investigation of mitochondrial epigenetics in canine oncology.