Abstract Thin interlayers are essential for high-quality multilayer optics. We present the first investigation of reducing the interlayer thickness of Mo/Si multilayer structures by cooling the substrate with liquid nitrogen during the deposition. The structures were deposited by means of electron beam evaporation. Even after warming up to room temperature prior to analysis, the interlayers that formed upon cryogenic deposition were found to be approximately 60% thinner compared to room temperature deposition. The interlayer thickness reduction at low temperature and its preservation upon warming up are attributed to a lower mobility of adatoms, reduced surface segregation of Si during Mo-on-Si growth, and/or crystallization of Mo.