Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long-term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non-canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma-bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression-free survival (P = 0.4) or overall survival (P = 0.1).