A comparative echocardiographic study was carried out on five shark species that differ in heart morphology and in aspects of their behavior and natural history. The study contrasted the ventricular function in the highly active mako shark (heart type IV) and four other sharks (heart type III) that differ in activity levels (i.e. the sedentary horn and swell sharks vs. the moderately active blue and smooth-hound sharks). All five species exhibited biphasic ventricular filling characterized by an early (conduit) and late (atrial systole) phase. In the mako shark, early filling was dominant as indicated by a higher early flow peak velocity, a greater early:late velocity ratio, and a greater early velocity time integral. In contrast, the late filling phase was the more important filling agent in the other species. Indices of systolic function such as ventricular ejection fraction and ventricular fractional shortening also reflect a more efficient cardiac pumping capacity in mako shark relative to the other four sharks. The comparative echocardiographic assessment of in vivo ventricular function integrates structural and functional features with shark activity level to arrive at a new perspective blending the occurrence of biphasic filling with functional concepts based on heart morphological typology and changing views regarding the role of factors such as central filling pressure and pericardial pressure on end-diastolic ventricular volume.