Pigmentation of murine cardiac tricuspid valve leaflet is associated with melanocyte concentration, which affects its stiffness. Owing to its biological and viscoelastic nature, estimation of the in situ stiffness measurement becomes a challenging task. Therefore, quasi-static and nanodynamic mechanical analysis of the leaflets of the mouse tricuspid valve is performed in the current work. The mechanical properties along the leaflet vary with the degree of pigmentation. Pigmented regions of the valve leaflet that contain melanocytes displayed higher storage modulus (7-10 GPa) than non-pigmented areas (2.5-4 GPa). These results suggest that the presence of melanocytes affects the viscoelastic properties of the mouse atrioventricular valves and are important for their proper functioning in the organism.