Objective-To evaluate molecular abnormalities in the c-kit gene of canine mast cell tumors (MCT) with different grades of cellular differentiation. Sample Population-31 normal tissue specimens from dogs and 45 canine MCT classified according to grade of cell differentiation. Procedure-Genomic DNA extractions were made from canine MCT and normal tissues. Parts of exon 11, intron 11, and exon 12 of the c-kit gene were amplified by use of polymerase chain reaction. These regions were cloned, sequenced, and compared with GenBank sequences of the National Center for Biotechnology International. A statistical analysis was used to compare sequences from canine MCT and normal tissues. Results-A significantly higher percentage of homozygous intron 11 deletion was found in canine MCT (49%) than in normal tissues (13%). This percentage was also higher in moderately and poorly differentiated MCT, compared with well-differentiated MCT. Although no mutations were detected in any of the specimens, a polymorphism at amino acid position 606 of the canine c-kit sequence was found in all the studied sequences. Conclusion and Clinical Relevance-Results indicated a relationship between intron 11 deletion and MCT, and the grade of MCT differentiation. We suggest that intron 11 deletion may be implicated in the pathogenesis of MCT and could be used as a marker for diagnosis and prognosis of canine MCT.