Our aim was (1) to determine the within-day and between-day variability of several indices of systolic and diastolic right ventricular (RV) function using conventional echocardiography and speckle tracking echocardiography (STE) (Study 1), (2) to quantify these variables in a large healthy canine population (n=104) with Doppler-derived estimated systolic pulmonary arterial pressure (SPAP) and left ventricular (LV) function, and (3) to establish the corresponding reference intervals (Study 2). For both studies, RV variables included tricuspid annular plane systolic excursion (TAPSE), fractional area change (RFAC), STE longitudinal systolic strain (StS) of the RV free wall (RVFW) and of the entire RV (i.e., global RV StS), STE longitudinal systolic RVFW strain rate (SRS) and the diastolic early:late SR ratio. All within- and between-day coefficients of variation, except one, (13/14) were <15%, the lowest being observed for TAPSE (3.6-9.8%), global RV (3.8-9.9%) and RVFW StS (3.7-7.3%). SPAP was weakly and negatively correlated with the TAPSE:body weight ratio (rs=-0.26, P=0.01) and RVFW SRS (rs=-0.23, p<0.05). Reference intervals (lower and upper limits with 90% confidence intervals) were provided for all variables. STE provides a non-invasive evaluation of RV function that may be used for clinical investigations in canine cardiology.