Cardiac myofibre deformation is an important determinant of the mechanical function of the heart. Quantification of myofibre strain relies on 3D measurements of ventricular wall motion interpreted with respect to the tissue microstructure. In this study, we estimated in vivo myofibre strain using 3D structural and functional atlases of the human heart. A finite element modelling framework was developed to incorporate myofibre orientations of the left ventricle (LV) extracted from 7 explanted normal human hearts imaged ex vivo with diffusion tensor magnetic resonance imaging (DTMRI) and kinematic measurements from 7 normal volunteers imaged in vivo with tagged MRI. Myofibre strain was extracted from the DTMRI and 3D strain from the tagged MRI. We investigated: i) the spatio-temporal variation of myofibre strain throughout the cardiac cycle; ii) the sensitivity of myofibre strain estimates to the variation in myofibre angle between individuals; and iii) the sensitivity of myofibre strain estimates to variations in wall motion between individuals. Our analysis results indicate that end systolic (ES) myofibre strain is approximately homogeneous throughout the entire LV, irrespective of the inter-individual variation in myofibre orientation. Additionally, inter-subject variability in myofibre orientations has greater effect on the variabilities in myofibre strain estimates than the ventricular wall motions. This study provided the first quantitative evidence of homogeneity of ES myofibre strain using minimally-invasive medical images of the human heart and demonstrated that image-based modelling framework can provide detailed insight to the mechanical behaviour of the myofibres, which may be used as a biomarker for cardiac diseases that affect cardiac mechanics.