Objectives - Two-dimensional (2D) speckle tracking echocardiography (STE) is a new angle-independent ultrasound technique based on tracking of speckles within the myocardium on 2D grayscale images. The aims of this prospective study were as follows: (1) to assess the variability of left ventricular peak systolic radial strain (St) and strain rate (SR) in awake dogs using STE (Protocol 1); and (2) to quantify these variables in a healthy canine population and compare them with tissue Doppler imaging (TDI)-based St and SR values (Protocol 2). Background - St and SR may be assessed using TDI, which is limited by angle dependency. Animals, materials and methods - Thirty-six STE examinations were performed on 6 healthy dogs for Protocol 1 and 37 healthy dogs were recruited for Protocol 2. In both studies, STE measurements were obtained offline from the right parasternal short-axis view by the same trained observer using automatic frame-to-frame tracking of grayscale speckle patterns. Results - All within- and between-day coefficients of variation were <10% (Protocol 1). In Protocol 2, St (46.7+or-12.2%) and SR (2.7+or-0.6 s-1) measured by STE were correlated with heart rate (p<0.01), but not with the ratio of early mitral inflow velocity to early mitral annular velocity. There was a good correlation between STE and TDI for both St and SR values (p<0.001). Conclusions - STE is a repeatable and reproducible non-Doppler method for assessing radial St and SR. The combination of these indices with conventional echo-Doppler variables could provide a new approach for accurately quantifying canine systolic function.