An image sensor includes a substrate, and a pixel separation pattern disposed in the substrate and interposed between a plurality of unit pixels. The plurality of unit pixels include a first unit pixel region and a second unit pixel region adjacent to the first unit pixel region in a first direction. The first unit pixel region and the second unit pixel region respectively include a first transfer gate and a second transfer gate. The pixel separation pattern includes a first pixel separation part interposed between the first unit pixel region and the second unit pixel region, and a second pixel separation part spaced apart from the first pixel separation part in the first direction. A top surface of the first pixel separation part is lower than a top surface of the second pixel separation part.