A retrospective study of military working dogs (MWDs) that were on active duty within the Department of Defense from 2000 to 2023 was conducted for the prevalence of osteosarcoma (OSA), histological subtypes and metastatic potential, and to examine potential risk factors. The prevalence of OSA in privately owned dogs (ie, pets of Department of Defense beneficiaries) that were patients of veterinary treatment facilities from 2000 to 2023 was used as a control. Between 2000 and 2023, 2,220 deceased MWD records had pathology data available for review, 27 of which had OSA (1.2% prevalence). The age at death was recorded in 22 of the 27 cases. During the same period, tissue specimens submitted to the Joint Pathology Center from privately owned dogs had pathology data available for review, and 83 were diagnosed with OSA. Breed, sex, neuter status, age at death and procurement location did not significantly affect the odds of an OSA diagnosis. MWDs that had patrol certification had decreased odds of OSA (odds ratio 0.37 with a 95% confidence interval [CI] of 0.17-0.78). When privately owned dogs were diagnosed with OSA they were unlikely to have axial OSA (22%). The corresponding figure in MWDs was much higher (45%). The occurrence of appendicular OSA was similar between MWDs (48%) and privately owned dogs (54%).