A metrology target formed by a lithographic process on a substrate includes a plurality of component gratings. Images of the target are formed using +1 and −1 orders of radiation diffracted by the component gratings. Regions of interest (ROIs) in the detected image are identified corresponding the component gratings. Intensity values within each ROI are processed and compared between images, to obtain a measurement of asymmetry and hence overlay error. Separation zones are formed between the component gratings and design so as to provide dark regions in the image. In an embodiment, the ROIs are selected with their boundaries falling within the image regions corresponding to the separation zones. By this measure, the asymmetry measurement is made more tolerant of variations in the position of the ROI. The dark regions also assist in recognition of the target in the images.