Elastic structure in cortical bone is usually simplified as orthotropic or transversely isotropic, which allows estimates of three-dimensional technical constants from ultrasonic and density measurements. These elastic property estimates can then be used to study phenotypic changes in cortical bone structure and function, and to create finite element models of skeletal structures for studies of organismal variation and functional adaptation. This study examines assumptions of orthotropic or transversely isotropic material structure in cortical bone through the investigation of off-axis ultrasonic velocities in the cortical plane in 10 samples each from a human femur, mandible and cranium. Longitudinal ultrasonic velocities were measured twice through each bone sample by rotating the perimeter of each sample in 1 ° angular intervals between two ultrasonic transducers. The data were fit to sine curves f(x) = (A × sin(x + B) + C) and the goodness of fit was examined. All the data from the femur fit closely with the ideal sine curve model, and all three coefficients were similar among specimens, indicating similar elastic properties, anisotropies and orientations of the axes of maximum stiffness. Off-axis ultrasonic velocities in the mandible largely fit the sine curve model, although there were regional variations in the coefficients. Off-axis ultrasonic velocities from the cranial vault conformed to the sine curve model in some regions but not in others, which shows an irregular and complex pattern. We hypothesize that these variations in ultrasonic velocities reflect variations in the underlying bulk microstructure of the cortical bone, especially in the three-dimensional patterns of osteonal orientation and structure. Elastic property estimates made with ultrasonic techniques are likely valid in the femur and mandible; errors in estimates from cranial bone need to be evaluated regionally. Approximate orthotropic structure in bulk cortical bone specimens should be assessed if ultrasound is used to estimate three-dimensional elastic properties.