Echocardiography is a noninvasive diagnostic tool that can provide instantaneous information about cardiac function, but it is uncommonly used by veterinarians to assess reptilian patients. Echocardiograms were performed on 14 clinically healthy, adult prairie rattlesnakes (Crotalus viridis), and cardiac measurements were taken in a horizontal and vertical position. Cardiac parameters including ventricular volume in systole and diastole, as well as the diameter of the left atrium, pulmonary artery, and paired left and right aortic arches were obtained. No evidence of cardiac disease was noted in any of the study animals. Males had a greater percentage of ventricular volume change (VVC) than females in the vertical position (P = 0.043). The percentage of ventricular volume change was significantly lower in the horizontal compared with the vertical position (P = 0.032) and was not different by sex. For the short-axis views, the right atrial diameter and short-axis ventricular area in diastole and systole were significantly smaller in snakes in the vertical compared with the horizontal position. This study is the first to obtain echocardiographic measurements in North American vipers and adds to the understanding of techniques used to evaluate the cardiac function of these species.