Bone is a composite material with a hierarchical structure. Its strength depends on its structural and material properties. In the present study, Raman microspectroscopic and Imaging analyses were employed to study 12 osteons in tissue sections from the femoral midshaft of a healthy human female, with a spatial resolution of ∼1 μm. Spatial changes in amount of mineral and organic matrix, as well as the variation in the mineral content were determined, imaged, and plotted as a function of the polarization of incident light. The results showed that the prominent bands, such as ν₁PO₄ and amide I, commonly used for the determination of mineral and organic compositions, are quite sensitive to the orientation and the polarization direction of the incident light. On the other hand, bands such as amide III, ν₂PO₄ and ν₄PO₄ are less susceptible to the orientational effects. As a result, exclusive consideration of the ν₁PO₄ and amide I bands for the calculation of material properties might lead to erroneous conclusions. Amide III, ν₂PO₄ and ν₄PO₄ Raman bands should also be taken into consideration for compositional analysis of bone structures, especially ones with unknown orientational features. Moreover, the results of the present study demonstrate the versatility of the analytical technique, and provide insights into the organization of bone tissue at the ultrastructural level.