Amelogenesis imperfecta (AI) is an inherited tooth disorder with widely varying phenotypes. The aim of this study was to determine the microhardness and microstructure characteristics of the enamel in AI teeth. The AI phenotypes examined were hypoplastic (pitted and smooth form), hypomaturated, and hypocalcified. Six AI patients were diagnosed according to clinical characteristics. The microhardness of the enamel was measured on axial cuts of AI teeth acquired from the patients. The measurements were done on several sites from the enamel surface towards the dentine-enamel junction using the Vickers scale. Values of microhardness were compared to corresponding control teeth. The microstructure of AI enamel types was evaluated using scanning electron microscopy. The values of microhardness in pitted hypoplastic AI samples were, on average, lower compared to the control enamel and dropped markedly towards the dentine-enamel junction. The smooth hypoplastic enamel was not only extremely thin but also much softer than control enamel. The values for hypomaturated AI fluctuated, but the palatal sites were markedly softer than in the control tooth. Hypocalcified enamel was the softest, with values resembling those of dentin. Microstructural changes varied from altered orientation of enamel prisms in pitted hypoplastic AI to lack of normal prismatic structure and severe porosity in hypocalcified AI. The present results suggest different microhardness profiles and microstructures in each phenotype. Variations among phenotypes are expected with larger case selection in this genetically heterogeneous disease.