The dependence of the giant magnetoresistance effect (GMR) on the interface structure in Fe/Cr multilayers was studied by magnetoresistivity and Mössbauer spectroscopy. The Fe/Cr multilayers consisting of Fe(6 nm)+[Cr(1.1 nm)/Fe(3 nm)]60+Cr(1.1 nm) were deposited by dc magnetron sputtering. Samples were annealed for 1 h at temperatures ranging from 200 to 500 °C. The interface structure was characterized by conversion electron Mössbauer spectroscopy (CEMS). Various different Fe sites: the bulk, step and perfect interface positions were identified. The evolution of the fraction of Fe atoms in different environments vs annealing temperature revealed that annealing at 300 °C induces bulk and in-plane diffusion of atoms. Higher annealing temperature causes substantial roughening of the interface related to a strong bulk diffusion of atoms. Microstructural changes observed in the CEMS spectra correspond well to the GMR measurements which reveal an enhancement of magnetoresistivity in Fe/Cr multilayers annealed at about 300 °C and its decrease due to deterioration of the interface at higher annealing temperatures.