Heart myocardia are critical to the facilitation of heart pumping and blood circulating around the body. The biaxial mechanical testing of the left ventricle (LV) has been extensively utilised to build the computational model of the whole heart with little importance given to the unique mechanical properties of the right ventricle (RV) and cardiac septum (SPW). Most of those studies focussed on the LV of the heart and then applied the obtained characteristics with a few modifications to the right side of the heart. However, the assumption that the LV characteristics applies to the RV has been contested over time with the realisation that the right side of the heart possesses its own unique mechanical properties that are widely distinct from that of the left side of the heart. This paper evaluates the passive mechanical property differences in the three main walls of the rat heart based on biaxial tensile test data. Fifteen mature Wistar rats weighing 225 ± 25 g were euthanised by inhalation of 5% halothane. The hearts were excised after which all the top chambers comprising the two atria, pulmonary and vena cava trunks, aorta, and valves were all dissected out. Then, 5 × 5 mm sections from the middle of each wall were carefully dissected with a surgical knife to avoid overly pre-straining the specimens. The specimens were subjected to tensile testing. The elastic moduli, peak stresses in the toe region and stresses at 40% strain, anisotropy indices, as well as the stored strain energy in the toe and linear region of up to 40% strain were used for statistical significance tests. The main findings of this study are: (1) LV and SPW tissues have relatively shorter toe regions of 10–15% strain as compared to RV tissue, whose toe region extends up to twice as much as that; (2) LV tissues have a higher strain energy storage in the linear region despite being lower in stiffness than the RV; and (3) the SPW has the highest strain energy storage along both directions, which might be directly related to its high level of anisotropy. These findings, though for a specific animal species at similar age and around the same body mass, emphasise the importance of the application of wall-specific material parameters to obtain accurate ventricular hyperelastic models. The findings further enhance our understanding of the desired mechanical behaviour of the different ventricle walls.