We present a novel multilayer grating pattern with a sub-50-nm pitch for critical dimension–scanning electron microscope (CD-SEM) magnification calibration as an advanced version of the conventional 100-nm pitch grating reference. A 25-nm pitch grating reference is fabricated by multilayer deposition of alternating materials and then material-selective chemical etching of the polished cross-sectional surface. A line and space pattern with 25-nm pitch is easily resolved, and a high-contrast secondary electron image of the grating pattern is obtained under 1-kV acceleration voltage using the CD-SEM. The uniformity of the 25-nm pitch of the grating is <1 nm in three standard deviations of the mean. The line-edge roughness of the grating pattern is also <0.5 nm. Such a fine and uniform grating pattern will fulfill the requirements of a magnification calibration reference for a next-generation CD-SEM.