Most existing preclinical models for evaluating the biosafety and bone-regeneration efficacy of innovative bone substitute materials (BSMs) or tissue engineering (TE) constructs only consisted of a single-site defect and the anatomical locations of defect varied drastically. While the compelling evidence showed that the bone healing pattern is location-dependent, owing to developmental, structural, and functional differences of anatomical locations, this is particularly true for the craniofacial region. Taking this into account, the bone healing efficiency of a BSM shown at one anatomical defect location cannot ensure the same impact at another. This prompted us to develop, for the first time, a model of bilateral critical-sized defect (CSD) at two distinctly different locations (non-load-bearing parietal calvaria and load-bearing mandibular body) co-existing in one rabbit to reduce the number of animals needed and avoid the influence of interindividual variability and evaluation bias on comparisons. 24 healthy adult male New Zealand White rabbits were randomly assigned to a group, either control, autograft (considered the “gold standard”) or a clinically relevant BSM (biphasic calcium phosphate granules) (BCPg, Mastergraft®, Medronics). The full-thickness cylindrical calvarial defect (ø10 mm) on frontoparietal region and mandibular composite defect (ø11 mm) on the body of the mandible were created bilaterally using low-speed drilling with saline irrigation. The defect on one side was filled with autograft debris or BCPg, and the other side was no graft (empty). Following the euthanasia of animals at the predetermined intervals (4w and 12w), the defect zones were examined macroscopically and then sampled and processed for microcomputed tomography (microCT) and histological analysis. All surgeries went uneventfully, and all rabbits recovered slowly but steadily. No symptoms of infection or inflammation associated with the defect were observed during the experiment. At 4w and 12w, macroscopic views of all defect sites were clean without any signs of necrosis or abscess, and no intraoral communication was found. The analysis of microCT and histological findings showed the non-healing nature of the empty defect, thereby both calvaria and mandible CSDs can be validated. The study of the application of BCPg in this defect model highlighted good osteointegration and excellent osteoconductive properties but compromised the osteoinductive properties of this material (compared with autograft). To conclude, this novel double-site CSD model holds great promise in the application for preclinical evaluation of BSMs, TE construct, etc. With a reduced number of animals in use, and lower interindividual variability and evaluation bias for comparisons.