In a method of aligning a substrate, a first alignment mark and a second alignment mark in a first shot region on the substrate may be sequentially identified. The substrate may be primarily aligned using identified any one of the first alignment mark and the second alignment mark. A used alignment mark and an unused alignment mark during the primary alignment process of the first alignment mark and the second alignment mark in a second shot region on the substrate may be sequentially identified. The substrate may be secondarily aligned using identified any one of the used alignment mark and the unused alignment mark during the primary alignment process. Thus, a time for identifying the alignment mark may be reduced.