Disclosed is a method of determining an overlay error between two layers of a multiple layer sample. For a plurality of periodic targets that each have a first structure formed from a first layer and a second structure formed from a second layer of the sample, an optical system is employed to thereby measure an optical signal from each of the periodic targets. There are predefined offsets between the first and second structures. An overlay error is determined between the first and second structures by analyzing the measured optical signals from the periodic targets using a scatterometry overlay technique based on the predefined offsets.