Two-dimensional (2D) and Doppler echocardiography is considered the most practical diagnostic tool for antemortem evaluation of reptilian heart disease but has only been described in a limited number of chelonian species. Cardiac morphology and function were evaluated by ultrasonography in 35 clinically healthy, captive non-sedated European pond turtles (Emys orbicularis) using 2D mode, M mode, and pulsed-wave spectral Doppler. Turtles were placed in a temperature-controlled water tank (24–26°C, 75.2–78.8°F) for cardiac examination through the right cervicobrachial window using water as coupling agent. Each echocardiogram included the evaluation of 19 variables—including six in 2D mode, seven in M mode, and six in Doppler mode—and was performed in 5.64 min on average. Morphological and functional abnormalities were identified in eight turtles, including atrioventricular insufficiencies (n = 5), pericardial effusions (n = 4), increased velocity of the ventricular outflow tract (n = 1), and bradyarrhythmia (n = 2). Reference intervals could be established for the 19 parameters in the remaining individuals after excluding outliers (n = 25). Statistically significant positive correlations between weight and diastolic (0.61, P = 0.001) and systolic (0.46, P = 0.018) ventricular volumes, and diastolic ventricular diameter (0.35, P = 0.03) were identified. This study establishes an initial database for the evaluation of captive European pond turtle cardiovascular system by a simple and clinically applicable echocardiographic technique.