An image sensor may include a substrate including first and second surfaces opposite to each other and including a single crystalline layer, a first epitaxial layer, and a second epitaxial layer sequentially stacked from the second surface. The single crystalline layer and the second epitaxial layer may be doped with first impurities of a first conductivity type. The first epitaxial layer may be doped with second impurities of a second conductivity type. A pixel separation structure extends from the first surface to penetrate at least the second and first epitaxial layers and divides the substrate into a plurality of pixels. A transfer gate electrode extends from the first surface to penetrate the second epitaxial layer. A doping concentration of the first impurities doped in the single crystalline layer may be higher than that in the second epitaxial layer.