In the current study, we examined the types and frequency of KIT mutations in mast cell tumors from 191 dogs. Sequencing of reverse transcription-PCR products revealed alterations in 50 (26.2%) of the dogs. Most mutations were in exon 11 (n = 32), and of these, most were internal tandem duplications (n = 25) between residues 571 and 590. Within exon 11, there were two hotspots for mutations at codons 555-559 and 571-590. In addition, nine dogs had mutations in exon 8 and eight had mutations in exon 9. We selected the two most common mutants and two representative exon 11 mutants for further analysis. When expressed in Ba/F3 cells, they were constitutively tyrosine phosphorylated and induced growth factor-independent cell proliferation. AG1296, a tyrosine kinase inhibitor, dose dependently inhibited both the tyrosine phosphorylation of these mutants and their induction of growth factor-independent proliferation. This study shows that activating mutations in not only exon 11 but also exons 8 and 9 are common in canine mast cell tumors. These results also show that Ba/F3 cells can be used for the direct characterization of canine KIT mutants, eliminating the need to make equivalent mutations in the mouse or human genes.