Hydrothermal isometric tension (HIT) testing and high performance liquid chromatography (HPLC) were used to assess the molecular stability and crosslink population of collagen in the four valves of the adult bovine heart. Untreated and NaBH4-treated tissues under isometric tension were heated in a water bath to a 90°C isotherm that was sustained for 5 hours. The denaturation temperature (T_d), associated with hydrogen bond rupture and molecular stability, and the half-time of load decay (t_1/2), associated with peptide bond hydrolysis and intermolecular crosslinking, were calculated from acquired load/temperature/time data. An unpaired group of samples of the same population was biochemically assayed for the types and quantities of enzymatic crosslinks present. Tissues known to endure higher in vivo transvalvular pressures had lower Td values, suggesting that molecular stability is inversely related to in vivo loading. The treated inflow valves (mitral and tricuspid) had significantly lower t_1/2 values than did treated outflow valves (aortic and pulmonary) suggesting lower overall crosslinking in the inflow valves. Inflow valves were also found to fail during HIT testing significantly more often than outflow valves: also suggestive of a decreased crosslink population. Inflow valves may be remodelling at a faster rate and may be at an early state of molecular 'maturity' than outflow valves. At the molecular level, the thermal stability of collagen is associated with in vivo loading and may be influenced by the mature, aldimine-derived crosslink, histidinohydroxylysinonorleucine (HHL). We conclude the valves of the heart utilize differing, location-specific strategies to resist biomechanical fatigue loading.