To investigate the thermal stability of Mo/Si multilayers with different initial crystallinities of Mo layers, two kinds of Mo/Si multilayers were deposited by DC magnetron sputtering and annealed at 300°C and 400°C. The period thickness compactions of multilayers with crystalized and quasi-amorphous Mo layers were 0.15 nm and 0.30 nm at 300°C, respectively, and the stronger the crystallinity, the lower the extreme ultraviolet reflectivity loss. At 400°C, the period thickness compactions of multilayers with crystalized and quasi-amorphous Mo layers were 1.25 nm and 1.04 nm, respectively. It was shown that multilayers with a crystalized Mo layer had better thermal stability at 300°C but were less stable at 400°C than multilayers with a quasi-amorphous Mo layer. These changes in stability at 300°C and 400°C were due to the significant transition of the crystalline structure. The transition of the crystal structure leads to increased surface roughness, more interdiffusion, and compound formation.