The anisotropic elastic symmetry of osteonal bone reflects the ultrastructural organization of collagen fibrils and mineral crystals within the osteons as well as the lamellar microstructure. Until recently, reported values for bone's anisotropic elastic properties were limited in their interpretation by poor precision and resolution of measurement techniques. Here, we report measurements of bone anisotropy using high precision acoustic microscopy. The elastic properties of canine femoral bone specimens, taken from 23 femora, were measured at 10° increments from the long axis of the bone. Half of the bone specimens subsequently were demineralized in EDTA solution, the other half were decollagenized in sodium hypochlorite solution, and the acoustic measurements were repeated. We found the elastic symmetry of osteonal bone deviates significantly from orthotropic theory supporting the hypothesis that the lamellar microstructure forms a “rotated plywood” (Weiner and Traub, FASEB J 6:​879–885;​ 1992). The principal orientation of bone mineral was along the long axis of the bone, while bone collagen appeared to be aligned at a 30° angle to the long axis. The misalignment between the mineral and the collagen suggests that (1) a substantial percentage of the mineral is extrafibrillar, and (2) the alignment of extrafibrillar mineral is governed by external influences, e.g., mechanical stresses.