The study aimed to identify molecular alterations in the tRNA-Leu (UUR), compare the methylation profile of this gene in healthy dogs versus dogs with malignant tumours, and to analyse whether there is a link between the presence of these changes and tumorigenesis. This study included 74 samples obtained from 47 dogs. The experimental group included 19 individuals with various malignant tumors (blood, tumor tissue) and 8 bitches with carcinoma tubulo-papillare (tumor and healthy tissue), one of the most common cancers in these animals. Notably, this study represents the first-ever analysis of the canine tRNA-Leu (UUR) gene methylation profile. The control group comprised 20 individuals without signs of disease (blood) of varying ages and breeds. Sanger sequencing was used, followed by bioinformatic assessments of the obtained sequences. Thus, the assessments revealed two variations: the m.2683A>G polymorphism present in 81% of the samples from the experimental group and in 85% of the samples from control group, as well as the m.2678_2679insG polymorphism, present in 100% of both groups. Furthermore, no changes were detected in the methylation profile of the tRNA-Leu (UUR) in both tumour and healthy tissue samples from the selected dogs. The detailed study did not show any tendency between observed molecular changes and type of the tumour. Further study is needed to verify the occurrence of alternations to analyse the molecular background of the disease comprehensively.