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The crystal structure of ZnxCo0.5-xFe2.5O4, prepared by a solvothermal reaction method, showed cubic spinel structure with space group Fd-3 m based on Rietveld refinement. The lattice constant a0 increased linearly with the Zn concentration from to 0.5. Field emission scanning electron microscope (FE-SEM) measurements showed that the size of the monodispersed particles was around 300-400 nm. With increasing Zn concentration, the saturation magnetization increased from 80.3 to 109.7 emu/g, while the coercivity at 293 K decreased from 893 to 46 Oe, respectively. The magnetocrystalline anisotropy constants (K1) were determined as 1.62, 1.32, and 1.16×106 erg/cm3 for x= 0.25, and 0.5, respectively, based on the law of approach to saturations (LAS) method. We have investigated the cation distribution by Mössbauer spectroscopy, closely related to K1. We have analyzed the recorded Mössbauer spectra as 3 sets with six-lines of tetrahedral site, and octahedral B1 and B2 sites both at 4.2 and 293 K. From the isomer shift values, the valence states of A and B1 site were determined to be ferric (Fe3+), while that at B2 site to be ferrous (Fe2+). The corresponding area ratio of A site decreased from 40 to 30 % while that of B site increased from 60 to 70% as the Zn concentration changed from x = 0 to 0.5 both at 4.2 and 293 K. Here, the changes in the area ratios of A and B sites are due to the changes in the cation distributions at the A and B sites, being originating from the randomly substituted Zn ions in ZnxCo0.5-xFe2.5O4 microspheres.