To further understand the cellular and molecular mechanisms underlying cortical bone graft healing, we have developed a novel mouse femur model that permits quantitative and molecular analysis of structural bone graft healing. A 4 mm mid-diaphyseal femoral segment was removed and replaced by either immediate implantation of a fresh autograft, a frozen, genetically identical isograft or a frozen allograft from a different strain of mouse, which was secured with a 22-gauge metal intramedullary pin. Healing was evaluated by radiology, histomorphometry, and in situ hybridization. Autograft repair occurred by endochondral bone formation at the host-graft junction and by intramembranous bone formation along the length of the graft bed at 2 weeks, with maturation and remodeling apparent by 4 weeks. Bone repair in allografts and isografts completely relied on endochondral bone formation at the host-graft cortical junction, with absence of periosteal bone formation along the length of the graft, suggesting that live periosteal cells from the donor tissue are necessary for this response. This small animal model of structural bone grafting can be used to evaluate tissue-engineered allografts and novel bone graft substitutes using quantitative and molecularly defined outcome measures.