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The material properties of some bones are known to vary with anatomical location, orientation and position within the bone (e.g., cortical and trabecular bone). Details of the heterogeneity and anisotropy of bone is an important consideration for biomechanical studies that apply techniques such as finite element analysis, as the outcomes will be influenced by the choice of material properties used. Datasets detailing the regional variation of material properties in the bones of the skull are sparse, leaving many finite element analyses of skulls no choice but to employ homogeneous, isotropic material properties, often using data from a different species to the one under investigation. Due to the growing significance of investigating the cranial biomechanics of the rabbit in basic science and clinical research, this study used nanoindentation to measure the elastic modulus of cortical and trabecular bone throughout the skull. The elastic moduli of cortical bone measured in the mediolateral and ventrodorsal direction were found to decrease posteriorly through the skull, while it was evenly distributed when measured in the anteroposterior direction. Furthermore, statistical tests showed that the variation of elastic moduli between separate regions (anterior, middle and posterior) of the skull were significantly different in cortical bone, but was not in trabecular bone. Elastic moduli measured in different orthotropic planes were also significantly different, with the moduli measured in the mediolateral direction consistently lower than that measured in either the anteroposterior or ventrodorsal direction. These findings demonstrate the significance of regional and directional variation in cortical bone elastic modulus, and therefore material properties in finite element models of the skull, particularly those of the rabbit, should consider the heterogeneous and orthotropic properties of skull bone when possible.