The characterization of immortalized canine osteosarcoma (OS) cell lines used for research has historically been based on phenotypic features such as cellular morphology and expression of bone specific markers. With the increasing use of these cell lines to investigate novel therapeutic approaches prior toin vivotranslation, a much more detailed understanding regarding the genomic landscape of these lines is required to ensure accurate interpretation of findings. Here we report the first whole genome characterization of eight canine OS cell lines, including single nucleotide variants, copy number variants and other structural variants. Many alterations previously characterized in primary canine OS tissue were observed in these cell lines, includingTP53mutations,MYCcopy number gains, loss ofCDKN2A,PTEN,DLG2,MAGI2, andRB1and structural variants involvingSETD2,DLG2andDMD. These data provide a new framework for understanding how best to incorporatein vitrofindings generated using these cell lines into the design of future clinical studies involving dogs with spontaneous OS.