A method of measuring an overlay offset using a scanning electron microscope system includes: scanning an in-cell region, which includes a lower structure and an upper structure stacked in a sample, using a primary electron beam with a landing energy of at least 10 kV; detecting electrons emitted from the scanned in-cell region; and measuring an overlay offset with respect to overlapping patterns included in the in-cell region using an image of the in-cell region that is generated based on the detected electrons emitted from the scanned in-cell region.