Introduction: There is little literature on the subvalvular apparatus of the atrioventricular valves’ of the heart. This investigation aimed to compare mammalian atrioventricular valve subvalvular apparatus; in particular the number and characteristics of true chordae tendineae (TChT) and their tissue transition areas – proximally with the valve leaflets, and distally with the papillary muscles. Materials and Methods: Sheep, pig and bovine fresh hearts were dissected (n = 9). The subvalvular apparatus of the mitral and tricuspid valves were visualised. Each TChT origin was grouped and counted according to papillary muscle and valve, and compared within and across the species. Appropriate statistical analyses were then applied to identify any correlations. Histological examination of the transition areas was also performed. Results: The tricuspid valve had significantly more TChT than the mitral (p = 0.04). On comparison of the TChT counts in both valves across the species, there were no significant differences. An unexpected finding was the abrupt transition from chordae collagen to papillary muscle. Conclusion: The tricuspid valve is under less pressure than the mitral but is connected to significantly more TChT. We have shown no significant difference between the numbers of TChT for each papillary muscle in either mitral or tricupsid valves across the species. Veterinary teaching emphasises that there is no clinically significant difference at a gross morphology level between these species. This is the first study to report that there is also no significant difference at the subvalvular level, and this has direct translational relevance for bioprosthetic cardiac valve replacement.