Gastrointestinal stromal tumors (GISTs), leiomyomas, and leiomyosarcomas are common mesenchymal neoplasms in the gastrointestinal (GI) tract of dogs. As previously diagnosed smooth muscle tumors of the canine GI tract are increasingly reclassified as GISTs, it becomes important to identify additional criteria that may assist in the diagnosis of these neoplasms, provide prognostic information, and offer targets for therapy. Examination of cluster of differentiation (CD), molecule expression (such as KIT [CD117] and CD34) as well as gross, histologic, and immunohistochemical features (such as tumor size, tumor location, mitotic index, AgNOR, and Ki67 labeling) in human GISTs has revealed new and valuable prognostic, diagnostic, and therapeutic information. In this study, GISTs were examined for the gross, histologic, and immunohistochemical features listed above. Forty-nine cases of canine gastrointestinal mesenchymal neoplasms from the Animal Medical Center (New York, NY) were categorized as GISTs (KIT positive), leiomyosarcoma/leiomyoma (KIT negative, smooth muscle actin [SMA], and/or desmin positive), or other (KIT, SMA, and desmin negative). A proportion (55%) of canine cases previously diagnosed as smooth muscle tumors were reclassified as GISTs according to KIT immunoreactivity. Statistical correlations with survival data were not possible because of insufficient follow-up data. However, there was a significant difference between mitotic index, AgNOR, and Ki67 scores depending on the location of the tumor (small vs large intestine). This study represents the first time CD34 immunoreactivity has been demonstrated in canine GISTs.