An image sensor includes a first pixel separation structure in a substrate to separate pixels from each other. The first pixel separation structure includes a conductive layer therein. Moreover, the image sensor includes a wire layer that is spaced apart from the conductive layer on the substrate. A second pixel separation structure is adjacent to the first pixel separation structure in a first horizontal direction and is connected to a contact. The first and second pixel separation structures extend longitudinally in a second horizontal direction that is perpendicular to the first horizontal direction.