Condylar fracture of the third metacarpal bone (Mc3) is the commonest cause of racetrack fatality in Thoroughbred horses. Linear defects involving hyaline articular cartilage, articular calcified cartilage (ACC) and subchondral bone (SCB) have been associated with the fracture initiation site, which lies in the sagittal grooves of the Mc3 condyle. We discovered areas of thickened and abnormally-mineralised ACC in the sagittal grooves of several normal 18-month-old horses, at the same site that linear defects and condylar fracture occur in older Thoroughbreds and questioned whether this tissue had altered mechanical properties. We embedded bone slices in PMMA, prepared flat surfaces normal to the articular surface and studied ACC and SCB using combined quantitative backscattered electron scanning electron microscopy (qBSE) and nanoindentation testing: this allowed correlation of mineralisation density and tissue stiffness (E) at the micron scale. We studied both normal and affected grooves, and also normal condylar regions. Large arrays of indentations could be visualised as 2-dimensional maps of E with a limit to resolution of indentation spacing, which is much larger than qBSE pixel spacing. ACC was more highly mineralised but less stiff in early linear defects than in control regions, while subchondral bone was more highly mineralised and stiffer in specimens with early linear defects than those without. Thus both ACC and SCB mineralisation may be abnormal in a class of early linear defect in 18-month-old Thoroughbred horses, and this may possibly contribute to later fracture of the Mc3 condyle.