One-dimensional strain imaging has been shown to be angle dependent. To address this problem, a new methodology, 2D-strain, has become available. The aim of this study was to validate this methodology in an in vivo set-up against sonomicrometry. In five open chest sheep, ultrasound gray-scale images were acquired of the inferolateral wall from two different angles. The longitudinal and radial strain components were simultaneously extracted using the novel 2D-strain methodology. The extracted values were compared with sonomicrometry using Bland-Altman statistics and correlation coefficients. A good agreement was found for the longitudinal strain component, while, for the radial strain estimates, the accuracy was less. 2D-strain is a fast and accurate tool to assess longitudinal strain from apical views. Further improvements are needed for the method to be sufficiently accurate in estimating the deformation perpendicular or close to perpendicular to the ultrasound beam.