The dominant diffusing species in the formation of an amorphous interlayer between Yb thin films and crystalline Si substrates have been determined by a Mo cluster marker experiment. Metal thin films with multilayered structures were deposited on both (111) and (001)Si substrates in an ultrahigh vacuum electron beam evaporation system. The positions of the Mo cluster markers relative to the Si substrates, before and after heat treatment, were determined by high-resolution transmission electron microscopy and energy dispersive analysis of x-ray as well as autocorrelation function analysis. The displacement of the Mo cluster markers in the amorphous interlayer during the Yb–Si interdiffusion indicates that Si atoms constitute the dominant diffusing species during the growth of amorphous interlayer.