One of the utmost concerns for the horse racing industry is the catastrophic injuries to the fetlock joint. In Thoroughbreds, proximal sesamoid bone (PSB) fractures account for a substantial fraction of all fatal musculoskeletal injuries, but the underlying pathogenesis is still poorly understood. In this study, we characterized the tissue material properties of PSBs by Raman spectroscopy to determine how bone tissue material properties were associated with fracture, exercise history, and anatomic regions. The results demonstrated that 1) the Carbonate:Phosphate Ratio increased slightly with cumulative high-speed furlongs; 2) the flexor region had a lower Mineral:Matrix Ratio and AGEs and a higher Carbonate:Phosphate Ratio; 3) mineral and GAGs content differed regionally between fracture and control groups. These observations reveal relationships between regional differences of bone tissue material properties and fracture incidences, exercise history, and in vivo loading history, which provide insight into the potential pathophysiology of catastrophic PSB fractures and thus reduce fracture risk.