An overlay mark, an overlay measurement method using the same, and a manufacturing method of a semiconductor device using the same are provided. The overlay mark is for measuring an overlay based on an image, is configured to determine a relative misalignment between at least two pattern layers, and includes first to fourth overlay marks. The first overlay mark has a pair of first Moire patterns disposed on a center portion of the overlay mark. The second overlay mark has a pair of second Moire patterns disposed so as to face each other with the first Moire patterns interposed therebetween. The third overlay mark has a pair of third Moire patterns disposed on a first diagonal line with the first Moire patterns interposed therebetween. The fourth overlay mark has a pair of fourth Moire patterns disposed on a second diagonal line with the first Moire patterns interposed therebetween.