Cardiac fiber direction is an important factor determining the propagation of electrical activity, as well as the development of mechanical force. In this article, we imaged the ventricles of several species with special attention to the intraventricular septum to determine the functional consequences of septal fiber organization. First, we identified a dual-layer organization of the fiber orientation in the intraventricular septum of ex vivo sheep hearts using diffusion tensor imaging at high field MRI. To expand the scope of the results, we investigated the presence of a similar fiber organization in five mammalian species (rat, canine, pig, sheep, and human) and highlighted the continuity of the layer with the moderator band in large mammalian species. We implemented the measured septal fiber fields in three-dimensional electromechanical computer models to assess the impact of the fiber orientation. The downward fibers produced a diamond activation pattern superficially in the right ventricle. Electromechanically, there was very little change in pressure volume loops although the stress distribution was altered. In conclusion, we clarified that the right ventricular septum has a downwardly directed superficial layer in larger mammalian species, which can have modest effects on stress distribution. NEW & NOTEWORTHY A dual-layer organization of the fiber orientation in the intraventricular septum was identified in ex vivo hearts of large mammals. The RV septum has a downwardly directed superficial layer that is continuous with the moderator band. Electrically, it produced a diamond activation pattern. Electromechanically, little change in pressure volume loops were noticed but stress distribution was altered. Fiber distribution derived from diffusion tensor imaging should be considered for an accurate strain and stress analysis.< Réduire