Abstract The use of transthoracic echocardiography in dolphins has been limited so far owing to technical and anatomical specificities. Anatomic M-mode (AMM) is a postprocessing echocardiographic technique generating M-mode studies from two-dimensional (2D) cineloops independently of the ultrasound beam orientation. The aim of the present study was to determine the within-day (repeatability) and between-day (reproducibility) variability of AMM echocardiography in awake healthy bottlenose dolphins (BN, Tursiops truncatus). Four adult BN trained to lie in left recumbency at the water surface were involved in the protocol. A total of 96 echocardiographic examinations were performed on 4 different days by a trained observer examining each BN 6 times per day. Video clips of 2D left parasternal long-axis views showing the left ventricle (LV) ventrally and the aortic root dorsally were recorded at each examination and analyzed for AMM measurements in a random order. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). All examinations were interpretable allowing calculation of 10 AMM variables (i.e., end-diastolic and end-systolic ventral and dorsal LV myocardial wall thicknesses as well as LV and aortic diameters, mean aortic diameter, and LV shortening fraction). Most within- and between-day CV values (18/20) were <15%, the lowest being observed for the end-diastolic LV diameter (1.6%). In conclusion, AMM provides a simple non-invasive evaluation of heart morphology and function in the awake BN with good repeatability and reproducibility of the measurements. Further studies are required to determine the corresponding reference intervals.