We presented a magnetic force microscopy and vibrating sample magnetometer study of perpendicular anisotropy of [Co-t/Fe-3t]5 (t=2, 3, 4, 5 nm) multilayers prepared by direct current magnetron sputtering on glass and Si substrates. It was found that except for the specimens with t=2 nm, most specimens exhibited obvious weak perpendicular anisotropy. By analyzing the anisotropy field and the root-mean-square roughness of phase shift of magnetic force images, we compared the perpendicular anisotropy among the multilayers deposited on Si and glass substrates. As a result, it is concluded that the primary origin of the weak perpendicular anisotropy can be attributed to the compressive stress induced by the lattice mismatch between Fe and Co layers. (c) 2008 Elsevier B.V. All rights reserved.