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The current study evaluated rotational deformation of the equine left ventricle by two-dimensional speckle tracking on the chordal level (MV), the papillary muscle level (PM) and, for the first time, at the apical level (AP). Sixteen healthy horses were examined. Data was acquired on three consecutive days by two observers. A similar systolic rotational motion at the three myocardial levels was identified, however with different amplitudes. As seen from the base, the motion starts with an initial (or early systolic) clockwise rotation followed by a counterclockwise rotation. There was a significant difference between the rotational amplitudes at each level (p < 0.0001). An increase in left ventricular rotation amplitude from the chordal to the apical level was found. As seen from the base, there is an initial clockwise rotation (positive rotation), followed by a dominant counter-clockwise rotation (negative rotation) at each level. The coefficient of variation on the papillary muscle level (PM) was the lowest compared to the MV and AP scan level. In conclusion, the left ventricular rotational (LVrot) motion can be evaluated non-invasively using two-dimensional speckle tracking at three different scan levels. Further studies are necessary to confirm our results. Additionally, this method should be evaluated in horses with cardiac dysfunction, at rest and after exercise.