The lamellar bone's strength is mainly affected by the organization of its mineralized collagen fibers and material composition. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of ∼ 1–2 μm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which allowed us to separate out orientation and composition dependent Raman lines, depending on the polarization directions. This approach gives information about lamellar bone orientation and variation in bone composition. It is shown that the ν1 PO₄ to amide I ratio mainly displays lamellar bone orientation;​ and ν₂ PO₄ to amide III and CO₃ to ν₂ PO₄ ratios display variation in bone composition. The ν₂ PO₄ to amide III ratio is higher in the interstitial bone region, whereas the CO₃ to ν₂ PO₄ ratio has lower values in the same region. The present study provides fresh insights into the organization of a lamellar bone tissue from two orthogonal orientations.