X-strainTM is a novel technique that allows determination of myocardial strain (S) and strain rate (SR) for assessment of regional myocardial function that is angle independent. This makes it an ideal tool for the evaluation of the right ventricle (RV) as it rotates on its axis in a non-uniform manner when it contracts. The technique has not previously been applied to bovine echocardiography and may enable better evaluation of RV cardiovascular function in clinical patients with cor pulmonale, myocarditis or pericarditis. The aims of this study were to report normal values and assess repeatability for right ventricular myocardial tissue velocities (TV), S and SR in healthy adult dairy cows. Eight healthy adult Holstein Friesian cattle (656±11Kg) were recruited and examined on three consecutive days. Standard echocardiographic images were obtained and offline analysis of RV function was performed using three cardiac cycles of the right parasternal long axis projections optimised to show either the left ventricular outflow tract (LVOT) or left ventricle (LV) and using a short axis projection at the level of the mitral valve (MV). Basal, mid and apical regions of the right ventricular free wall and right, mid and left regions of the interventricular septum (IVS) were compared both on the same view and on comparable views (LVOT and LV view). Data, shown as mean (±SEM), were normally distributed and analysed using a repeated measures ANOVA and Student's T-test where appropriate. Where no significant differences were found between regions/projections, data were combined. Repeatability was determined using intra-class correlation coefficients (ICC). Measures of free wall function were not affected by region and were consistent between the two different long axis projections (TV 4.5±0.73cm/s, S -16±4.1% and SR -1.29±0.23 s-1). IVS S and SR were not affected by region (S -12±2.6% SR -1.25±0.61 s-1) although IVS TV differed between the regions assessed (basal 6.8±0.59cm/s, mid 3.81±0.88cm/s and apical 2.53±0.59 cm/s). All measures of IVS function were consistent between the two different long axis projections. With the exception of SR of the basal free wall all X-strain measures demonstrated good repeatability (ICC>0.85). This technique is repeatable and values are comparable for those reported for the human right ventricle. X-strain may provide a superior method for assessing RV function in cattle with different forms of cardiac disease compared to 2D echocardiography. Evaluation in the clinical setting is warranted.