An image sensor includes a pixel array and a logic circuit. The pixel array includes a pixel isolation layer between a plurality of pixels. Each of the plurality of pixels include a pixel circuit below at least one photodiode. The logic circuit acquires a pixel signal from the plurality of pixels. The pixel array includes at least one autofocusing pixel, which includes a first photodiode, a second photodiode, a pixel internal isolation layer between the first and second photodiodes, and a microlens on the first and second photodiodes. The pixel internal isolation layer includes a first pixel internal isolation layer and a second pixel internal isolation layer, separated from each other in a first direction, perpendicular to the upper surface of the substrate, and the first pixel internal isolation layer and the second pixel internal isolation layer include different materials.