Mast cell tumours are one of the most common neoplasms in dogs. Mutations in the proto-oncogene c-kit, especially internal tandem duplications of exon 11, are considered to play a crucial role in mast cell tumourigenesis. In this report, two cases that suffered from multiple mast cell tumours containing an internal tandem duplication in the primary lesion but not in the secondary lesions are described. This finding indicates the existence of heterogenous c-kit gene mutations in each site of multiple mast cell tumours. Additionally, these results raise the possibility that the contribution of internal tandem duplications in the malignant transformation of mast cells is quite limited. It is proposed that, for clinicians, genetic analysis of several regions of multiple mast cell tumours is necessary for predicting prognosis and tumour response to KIT inhibitors.