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Simple Summary: Fatal injuries in Thoroughbred racehorses typically occur due to an accumulation of bone damage, however, detecting their impending onset can be difficult as there are often no overt signs. In other horse populations, facial grimacing has been shown to be associated with orthopaedic pain. This study, therefore, aimed to investigate facial expressions of Thoroughbred racehorses at the trot to identify if there were subtle signs of pain in mildly lame compared to non-lame horses. Two independent observers assessed 22 facial expression parameters using still photographs of the head from video-recorded trot-ups. There were few expressions associated with lameness except for more exposed whites of the eye in lame horses. Thus, facial pain scoring may not be adequate for the detection of subtle lameness in racehorses who work in a racing stable environment. Musculoskeletal injuries in racehorses are difficult to detect prior to catastrophic breakdown. Lameness is commonly attributed to orthopaedic pain in horses, therefore, subtle lameness may be a pre-clinical sign of injury and, if identified early, could allow for preventative intervention. Our objective was to determine if facial expressions could be used to detect mild lameness as an indicator of orthopaedic pain in 'fit to race' horses. The Horse Grimace Scale (HGS) and the facial expressions in ridden horses (FEReq), were used to score images (n = 380) of mildly lame (n = 21) and non-lame (n = 17) Thoroughbred horses by two independent observers. Using an Equinosis Lameness Locator®, the lameness status of each horse was determined according to published thresholds [forelimb lameness (>|14.5 mm|) and hindlimb lameness (>|7.5 mm|)]. Inter and intraobserver reliability were assessed using two-way random-effects models. Univariable associations between lameness and facial expression parameters were identified using logistic and linear regression. Interobserver reliability was moderate (κ 0.45; 95% CI 0.36, 0.55). Horses with moderate mouth strain (HGS) and tense and extended upper lip (FEReq) were less likely to be lame (p = 0.042 and p = 0.027, respectively). Exposed sclera was associated with lameness (p = 0.045). Higher orbital tightening (HGS) scores were associated with a lower degree of maximum head amplitude (HDmax) lameness (p = 0.044). Tension and moderate tension above the eye, for the HGS and FEReq scores, were associated with increasing amplitude of HDmax (p = 0.048 and p = 0.034, respectively). Inconsistent associations between lameness status and HGS and FEReq scores may limit the potential use of the facial expression for the prediction of mild orthopaedic pain during pre-race lameness examinations. More objective parameters associated with mild orthopaedic pain should be explored. [ABSTRACT FROM AUTHOR]