Disclosed is a method of measuring overlay between upper and lower layers on a substrate using metrology targets formed by a lithographic process. The lithographic process is of a multiple-patterning type whereby first and second distinct populations of structures are formed in a single one of said layers (L1) by respective first and second patterning steps. The metrology target (620) in the single one of said layers comprises a set of structures of which different subsets (642A, 642B) are formed in said first and second patterning steps. An overlay measurement on this target can be used to calculate a combined (average) overlay performance parameter for both of the first and second patterning steps.