Accurate clinical prediction of the resistance to fracture after callus distraction requires a detailed understanding of structural and elastic properties of the newly formed bone. We investigated 26 sheep that underwent middiaphyseal callus distraction at a rate of 0.5 mm every 12 h for 30 d using a standard unilateral fixator system. The sample population included four groups undergoing different treatments to improve bone healing, including bone grafting and the local application of growth factors. All animals were sacrificed eight weeks after the end of distraction. The fracture forces of the lengthened tibia and the contralateral control tibia from each animal were evaluated by biomechanical (four-point bending) testing. The microstructure and anisotropic acoustic impedance distributions were assessed by quantitative 50-MHz scanning acoustic microscopy. The relationships between resistance to fracture, structural properties and acoustic impedance of the newly formed callus tissue and adjacent cortical tissue were investigated. A significant linear multivariate regression model was developed that predicts the fracture force with a high accuracy (RMSE = 248 N, R(2) = 0.86, p < 0.0001).