We present optimization of [(15Å) Ni80Fe20/(5Å) M]20 single crystal multilayers on (001) MgO substrates, with M being Cu, Cu50Pt50 and Pt. These superlattices were characterized by high resolution X-ray reflectivity (XRR) and diffraction (XRD) as well as polar mapping of important crystal planes. It is shown that cube on cube epitaxial relationship can be obtained when depositing at substrate temperature of 100 °C regardless of the lattice mismatch (5% and 14% for Cu and Pt, respectively). At lower substrate temperatures poly-crystalline multilayers were obtained while at higher substrate temperatures {111} planes appear at ∼10° off normal to the film plane. It is also shown that as the epitaxial strain increases, the easy magnetization axis rotates towards the direction that previously was assumed to be harder, i.e. from [110] to [100], and eventually further increase in the strain makes the magnetic hysteresis loops isotropic in the film plane. Higher epitaxial strain is also accompanied with increased coercivity values. Thus, the effect of epitaxial strain on the magnetocrystalline anisotropy is much larger than what was observed previously in similar, but polycrystalline samples with uniaxial anisotropy (Kateb et al. 2021).