Canine Mast cell tumors (MCTs) constitute approximately 21% of all canine skin tumors. For intermediate graded MCTs, biological aggressiveness is difficult to predict. Progression in various cancers involves DNA hypermethylation, hypomethylation and epigenetic enzyme dysregulation. Therefore, levels of 5-methylcytosine, 5-hydroxymethylcytosine and associated enzyme expression may predict MCT aggressiveness. A tissue microarray with cores from 244 different tumor samples from 189 dogs was immunolabelled. H-scores were generated using QuPath (v0.1.2) and analyzed with associated patient data. High 5MC, DNMT1, and DNMT3a and low TET2, IDH1, and IDH2 levels were associated with poorer outcome in all canine MCT cases. High 5MC showed the most significance when stratifying cases by dermal and subcutaneous tissue location, and in intermediately graded cases. Therefore, there is potential for these markers to be able to predict more aggressive behavior and identify aggressive cases based on location as well as in intermediately graded MCTs.