Background: Echocardiography is commonly used for assessing cardiac structure and function in various species including non-human primates. A few previous studies reported normal echocardiographic reference intervals of clinically healthy rhesus macaques under sedation. However, these studies were under-powered, and the techniques were not standardized. In addition, body weight, age, and sex matched reference intervals should be established as echocardiographic measurements are commonly influenced by these variables. The purpose of this study was to establish reference intervals for complete echocardiographic parameters based on a large cohort of clinically healthy rhesus macaques with wide ranges of weight and age distributions using allometric scaling. Results: A total of 823 rhesus macaques (ages 6 months to 31 years old; body weights 1.4 to 22.6 kg) were enrolled. Of these rhesus macaques, 421 were males and 402 were females. They were assessed with a complete echocardiographic examination including structural and functional evaluation under sedation with ketamine hydrochloride. The reference intervals of the key echocardiographic parameters were indexed to weight, age, and sex by calculating the coefficients of the allometric equation Y = aMb. On correlation matrix, body weight, age, sex, and heart rate were significantly correlated with various echocardiographic parameters and some of the parameters were strongly correlated with body weight and age. Multiple regression analysis was also performed to predict various echocardiographic parameters from heart rate, body weight, age and sex. Heart rate and body weight statistically significantly predicted various echocardiographic parameters. Valve regurgitation including tricuspid, aortic, pulmonic, and mitral regurgitations without other cardiac structural and functional abnormalities are common in clinically healthy rhesus macaques under ketamine sedation. Conclusions: In this study, the reference intervals of echocardiographic parameters were established by performing complete echocardiographic examinations on a large number of clinical healthy rhesus macaques. In addition, allometric scaling was performed based on their weight, and further indexed to age and sex. These allometrically scaled reference intervals can be used to accurately evaluate echocardiographic data in rhesus macaques and diagnose structural and functional evidence of cardiac disease.