Scanning X-ray nanodiffraction on a highly periodic ferroelectric domain pattern of a strained K0.75Na0.25NbO3 epitaxial layer has been performed by using a focused X-ray beam of about 100 14;nm probe size. A 90°-rotated domain variant which is aligned along [1 2]TSO has been found in addition to the predominant domain variant where the domains are aligned along the [12]TSO direction of the underlying (110) TbScO3 (TSO) orthorhombic substrate. Owing to the larger elastic strain energy density, the 90°-rotated domains appear with significantly reduced probability. Furthermore, the 90°-rotated variant shows a larger vertical lattice spacing than the 0°-rotated domain variant. Calculations based on linear elasticity theory substantiate that this difference is caused by the elastic anisotropy of the K0.75Na0.25NbO3 epitaxial layer