Regional odontodysplasia (ROD), a rare developmental odontogenic defect severely compromising oral health and quality of life, was investigated through integrated microstructural, compositional, and biomechanical analyses of two exfoliated primary teeth compared to caries-free controls. Clinical examinations revealed yellowish discoloration, surface roughness, and advanced root resorption, while radiographs demonstrated hypocalcified enamel, enlarged pulp chambers, and delayed permanent successor development. Micro-computed tomography (micro-CT) demonstrated marked enamel thinning and reduced mineral density in both enamel and dentin . X-ray diffraction (XRD) analysis confirmed compromised hydroxyapatite crystallinity in ROD enamel, with lattice expansion along the a-axis. Ultrastructural defects were evident via scanning electron microscopy (SEM) and transmission electron microscopy (TEM), showing hypomineralized enamel prisms with irregular cross-banding and disorganized dentin collagen matrices with aberrant calcospherite fusion. Energy-dispersive X-ray spectroscopy (EDS) quantification revealed significant elemental deviations: Ca/P ratio reduction and Mg/Ca ratio elevation. Nanoindentation evidenced severe biomechanical compromise, with ROD enamel hardness and elastic modulus reduced. These multiscale alterations highlight the critical need for early interventions targeting structural reinforcement and permanent dentition monitoring to mitigate ROD-associated complications.