Abstract: Background – The size of the ascending aorta (aortic root) is of clinical relevance, in particular in cats with systemic hypertension and as a variable of the left atrium-to-aortic ratio, used in the echocardiographic diagnosis of left atrial enlargement. Hypothesis/Objectives – Determine reference values and 95% prediction intervals for two-dimensional echocardiographic variables of aortic root size in cats. We hypothesized that dimensions of the aortic root in healthy cats are mainly dependent on body weight and age. Animals – 220 healthy cats. Methods – Retrospective observational study. Systolic and diastolic dimensions of the aortic valve, the sinus of Valsalva, the sinotubular junction, and the ascending aorta and sinus height were evaluated. Reference values and 95% prediction intervals were determined using raw data and values after allometric transformation. The effect of body weight and age was determined using multivariable regression analysis. Results – Proportionality constants (), 95% prediction intervals, and allometric scaling exponents (b) were computed for all variables and reference values and prediction intervals determined. Body weight and age, but not sex and breed, were independent predictors of aortic root size. Cats >12 years old had significantly (adjusted P < .05) increased echocardiographic variables of aortic root size compared to cats 1-2 years old, >2-6 years old, and >6-12 years old. Conclusions and clinical importance – Reference and prediction intervals of echocardiographic variables characterizing aortic root size in cats are provided. The effect of body weight and age has to be considered for clinical decision-making when using these variables in clinical practice.