The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.