Myocardial stiffness is a novel biomarker with both diagnostic and prognostic potential in a range of cardiac diseases such as ischemia or myocardial infarction known to have increased stiffness. Application of Magnetic Resonance Elastography (MRE) to the heart enables measurement of myocardial stiffness in vivo. This study was performed to measure in-vivo myocardial stiffness during systole and diastole in pigs (n=17) using 3D MRE. A custom passive driver was placed on the chest and imaging was performed in prone position on a 1.5 Tesla whole body MR imager (Signa Excite; GE) with a 4-channel coil in oblique plane using ECG-gated spin-echo echo planar imaging sequence at 140 Hz vibration frequency with 5 breath holds of approximately 25 seconds. Systolic and diastolic short-axis acquisition was performed prescribing corresponding time delays observed from a FIESTA cine scan. Acquisition parameters: 1 shot, NEX = 1; TR/TE = 4600/52ms; FOV = 28.8 cm; 96x96 image matrix; 11 continuous 3 mm thick slices with 0 mm spacing, isotropic acquisition; 2 motion-encoding gradient (MEG) pairs; x, y, and z motion-encoding directions; ASSET= 2, and 4 phase offsets. MRE stiffness was obtained using 3D Local Frequency Estimation (LFE) algorithm and a ROI covering the left ventricle was used to report stiffness. The mean stiffness of the myocardium in systole was 5.89±0.34 kPa which was significantly higher (p < 0.01) than diastolic stiffness 3.91 ±0.39 kPa across 17 pigs. The results motivate in-vivo assessment of myocardial stiffness throughout the cardiac cycle and also provide a reference value at 140 Hz vibration frequency to develop pig models with cardiac diseases such as heart failure with preserved ejection fraction (HFpEF) that is known to have increased global stiffness.