The objectives of this study were (1) to assess the potential effect of body weight (BW), age, and gender on the most commonly used echocardiographic and conventional Doppler variables in a large population of healthy Cavalier King Charles Spaniels (CKCS), and (2) to establish the corresponding reference intervals (RI). 134 healthy adult CKCS. Ultrasound examinations were performed by trained observers in awake dogs. M-mode variables included left ventricular (LV) end-diastolic and end-systolic diameters, LV free wall and interventricular septal thicknesses at end-diastole and end-systole, and LV fractional shortening (FS%). The left atrium (LA) and aortic (Ao) diameters were measured using a 2D method, and the LA/Ao was calculated. Pulsed-wave Doppler variables included peak systolic aortic and pulmonary flow velocities, mitral E and A waves, and E/A ratio. Effects of BW, age, and gender on these 15 variables were tested using a general linear model, and RIs were determined by applying the statistical procedures recommended by the Clinical and Laboratory Standards Institute. A significant BW effect was observed for all variables, except LA/Ao, FS%, and mitral E/A ratio. A significant but negligible effect of gender and age was also observed for 5/15 and 4/15 of the tested variables, respectively. Only the BW effect on M-mode variables was considered as clinically relevant and the corresponding regression-based RIs were calculated Body weight should be taken into account when interpreting echocardiographic values in CKCS, except for LA/Ao, FS%, and mitral E/A ratio