The emergence of the four-chambered heart and its underlying substructures was a critical event in vertebrate evolution, appearing at least twice independently; once in the vertebrate group that gave rise to crocodilians and birds (archosaurs), and once in the ancestral group of mammals (synapsids). A fully divided ventricle provides for the ability to maintain high cardiac outputs and systemic arterial blood pressures, while simultaneously keeping low blood pressures within the pulmonary circulation. Low pulmonary blood pressures allow for a thinner blood-gas barrier within the lungs, an important component of overall higher pulmonary diffusion capacity and hence more efficient gas exchange. In addition, a fully divided ventricle also avoids the mixture of oxygen-rich and oxygen-poor blood within the heart. These characteristics are both necessary for supporting increased rates of metabolism, which is directly linked with the rapid expansion and success of mammals and birds (Hicks and Wang, 2012). Inferences about cardiac evolution result from studies investigating comparative cardiac morphology and function in extant species. Although extant reptiles (turtles, lizards, snakes and crocodilians) are only distantly related to modern mammals and birds, this clade provides the best group of animals for investigating and ultimately understanding the evolution of the four-chambered heart. The heart of non-crocodilian reptiles has two anatomically divided atria, and a single ventricle. Between the many species of reptiles, the internal morphology of the ventricle exhibits a large degree of variability, with many folds and septae. Regardless of the anatomical variability within the ventricle, a functional characteristic of the heart within all non-crocodilian reptiles is the potential for the mixing of oxygen rich blood and oxygen poor blood (cardiac shunting). Cardiac shunting is defined by its directionality, with right-to-left shunts (R-L) being defined as a portion of systemic venous blood bypassing the lungs and reentering the systemic circulation. Alternatively, a left-to-right (L-R) shunt represents pulmonary venous blood (oxygen rich) recirculating back to the lungs. Emerging from the single ventricle are the three great vessels; the pulmonary artery, the left aortic arch (LAo) and right aortic arch (RAo) (Hicks and Wang, 2012). It is interesting to note that crocodilians evolved a four-chambered heart, similar to birds and mammals. However, even in these animals, the separation of the pulmonary and systemic circulations is not absolute. Crocodilians retain the ancient phenotype of dual aortae (LAo and RAo), with the LAo emerging from the right ventricle, along with the pulmonary artery. The RAo exits the left ventricle. This unique anatomical arrangement results in the capacity for portion of the systemic venous blood to bypass the lungs and reenter the systemic circulation (right-to-left shunt) (Hicks and Wang, 2012). In the latter half of the 20th century, many comparative cardiovascular physiologists argued that cardiac shunts were an adaptive trait in non-avian reptiles. Basically it was reasoned; why would reptiles maintain such a unique cardiac morphology and shunting capacity if such traits were not adaptive? This view resulted in a number of hypotheses about the adaptive advantages of cardiac shunting (see Hicks and Wang, 2012 for review). However experimental support for an adaptive significance of cardiac shunting in non-avian reptiles has been lacking. For physiological or morphological trait to be considered adaptive it is important to provide evidence that the absence of the trait actually reduces physiological performance and/or reproductive fitness. Lacking such evidence, it is just as likely, that the unique features and functions of the reptilian heart is simply an embryonic or ancestral character; a character that does not negatively impact overall animal fitness and therefore has not been selected against. However, quantifying genetic fitness in reptiles is nearly impossible. Consequently the “adaptiveness” of cardiac shunts and determining the factors that drove the evolution of the four chambered heart and its characteristic structures, must be inferred by using a strategy that takes advantage of a variety of analytical and experimental approaches. Several of these approaches will be discussed in this symposium.