Currently, the molecular background based on mitochondrial DNA (mtDNA) analysis of canine testicular tumours is underestimated. The available data mostly focus on histopathological evaluations, with a few reports of nuclear genome (nDNA) studies. Tumourigenesis represents a highly complex and diverse genetic disorder, which can also encompass defects in mtDNA. The aim of this study was to identify molecular changes in whole mitochondrial genome sequences obtained from dogs affected by testicular tumours. Samples of blood, tumour, and healthy tissue were collected from each animal, and mtDNA (ultimately 45 samples) was subsequently sequenced. Thereafter, protein analyses were performed to assess the impact of the identified molecular alterations on the amino acid level. The total number of observed changes included 722 SNPs, 12 mutations, 62 indels, 5 indel mutations, and 35 heteroplasmic sites. The highest number of mtDNA variants in protein-coding genes COX1, COX3, ATP6, ND1, ND4, and ND5 was observed. Interestingly, SNPs were found in 10 out of 22 tRNA genes. Most of the identified mtDNA defects were synonymous changes at the amino acid level. Also, polymorphisms and heteroplasmy were frequently observed in the variable number of tandem repeat (VNTR) regions, especially in its fragment spanning 16,138–16,358 bp. Based on the obtained results, it was possible to select 11 polymorphisms that occurred in all the tested samples (benign, malignant) and an additional five SNPs identified only in benign neoplasms. The comprehensive analysis of malignant testicular tumours demonstrated a significant diversity in their molecular profiles, with changes ranging from 17 to 101 per sample.