Musculoskeletal injury remains a major welfare and performance concern in Thor-oughbred racing, and practical biomarkers for early risk stratification are needed. This multicentre observational cohort study characterised serum concentrations of two bone turnover markers, osteocalcin/BGLAP (OC) and C-terminal telopeptide of type I collagen (CTX-I), in 1,359 fit-to-race Thoroughbred racehorses sampled across New South Wales, Victoria, and Western Australia. Biomarker distributions, reference intervals, demo-graphic and training-related associations, and short-term soundness outcomes were evaluated using non-parametric methods. Both markers were positively skewed, with reference intervals of 0.13–9.44 ng/mL for CTX-I and 0.02–7.25 ng/mL for OC. CTX-I varied significantly by age, jurisdiction, venue, and training surface, with markedly higher concentrations in New South Wales horses and lower concentrations associated with polytrack training, but did not predict lameness outcomes. OC was significantly higher in horses classified as lame at sampling and showed modest but significant dis-criminatory ability for subsequent lameness, with strongest performance for persistent lameness at both 7 and 28 days. At an operational threshold of approximately 1.24 ng/mL, OC achieved high negative predictive value for persistent lameness. These findings support OC as a potential rule-out screening marker for short-term soundness risk, while CTX-I appears more informative for population-level skeletal turnover variation.