A method for determining overlay between layers of a multilayer structure may include obtaining a given image representing the multilayer structure, obtaining expected images for layers of the multilayer structure, providing a combined expected image of the multilayer structure as a combination of the expected images of said layers, performing registration of the given image against the combined expected image, and providing segmentation of the given image, thereby producing a segmented image, and maps of the layers of said multilayered structure. The method may further include determining overlay between any two selected layers of the multilayer structure by processing the maps of the two selected layers together with the expected images of said two selected layers.