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X-ray diffraction analysis and orientation contrast scanning electron microscopy imaging of La0.7Sr0.3MnO3 epitaxial layers grown on (001)-SrTiO3 substrates have been used to track the shear strain and twin domain period as a function of the thickness of the films, t. To this end, the diffraction by a periodically modulated twinned structure is analyzed in detail. In contrast with current equilibrium models, here we demonstrate the occurrence of a critical thickness, tτ ∼ 2.0-2.5 nm, for twin formation in rhombohedral perovskite films. The absence of twinning below tτ is explained by the formation of a monoclinic interfacial phase presumably driven by electronic interactions between film and substrate not taken into account in theoretical models. Above tτ, twin domains develop concomitantly with the build-up of misfit shear strains associated with the formation of the rhombohedral structure. At a thickness ∼10 nm, the in-plane and out-of-plane shear strain components exhibit similar values, as imposed by the rhombohedral symmetry. However, upon increasing the film thickness, both strain components are found to follow divergent trajectories indicating a progressive perturbation of the octahedral framework which allows the in-plane lattice parameters to remain fully strained within the explored thickness range (up to 475 nm). Despite these structural perturbations, the twin size follows a t1/2 dependence as predicted for homogeneous films by equilibrium models. © 2013 The Royal Society of Chemistry., This research was supported by Spanish MEC (MAT2009-08024 and MAT2011-29081), CONSOLIDER (CSD2007-00041 and CSD2008-00023), and FEDER program. Z. K. thanks the Spanish MEC for the financial support through the RyC program.