Organoid cultures could constitute a valuable in vitro model to explore new treatments for canine (c) medullary thyroid carcinoma (MTC). The study's objectives were to establish and characterize 3D organoid cultures of cMTC using histology and immunohistochemistry (IHC) and to evaluate the effect of antitumor drugs on organoids' viability. Five cMTC tissue samples were used to develop organoid cultures of which one organoid line, named cMTC N°2, could be passaged for an extended period. This cMTC N°2 organoid line was further compared to the primary tumour regarding morphology and IHC expression of thyroid transcription factor-1 (TTF-1), thyroglobulin, calcitonin, synaptophysin, vimentin, Ki-67, cyclooxygenase-2 (COX-2), P-glycoprotein and vascular endothelial growth factor (VEGF). Quality control of the cMTC N°2 organoid line was achieved by a single nucleotide polymorphism (SNP) array of the organoids, primary tumour and healthy blood cells of the same dog. The effect of carboplatin, meloxicam and toceranib phosphate (TOC) on cMTC N°2 organoids' viability was evaluated. The cMTC N°2 organoid line was cultured for 94 days and showed similar histological features with the primary tumour. Immunolabelling for TTF-1, thyroglobulin, calcitonin and VEGF was similar between the primary tumour and cMTC N°2 organoids. Compared to the primary tumour, organoids showed higher immunolabelling for vimentin and Ki-67, and lower immunolabelling for synaptophysin, COX-2 and P-glycoprotein. The SNP genotype was similar for each chromosome between healthy blood cells, primary tumour and cMTC N°2 organoids. Carboplatin, meloxicam and TOC had no effect on cMTC N°2 organoid cell viability within achievable in vivo concentration range. In conclusion, the cMTC N°2 organoid line is a promising first milestone towards an established in vitro organoid model to explore pathophysiology and new treatment modalities in cMTC.