Tendons are collagenous soft tissues that transmit loads between muscles and bones. Depending on their anatomical function, tendons are classified as positional or energy-storing with differing biomechanical and biochemical properties. We recently demonstrated that during monotonic stretch of positional tendons, permanent denatured collagen begins accumulating upon departing the linear region of the stress-strain curve. However, it is unknown if this observation is true during mechanical overload of other types of tendons. Therefore, the purpose of this study was to investigate the onset of collagen denaturation relative to applied strain, and whether it differs between the two tendon types. Rat tail tendon (RTT) fascicles and rat flexor digitorum longus (FDL) tendons represented positional and energy-storing tendons, respectively. The samples were stretched to incremental levels of strain, then stained with fluorescently labeled collagen hybridizing peptides (CHPs);​ the CHP fluorescence was measured to quantify denatured collagen. Denatured collagen in both positional and energy-storing tendons began to increase at the yield strain, upon leaving the linear region of the stress-strain curve as the sample started to permanently deform. Despite significant differences between the two tendon types, it appears that collagen denaturation is initiated at tissue yield during monotonic stretch, and the fundamental mechanism of failure is the same for the two types of tendons. At tissue failure, positional tendons had double the percentage of denatured collagen compared to energy-storing tendons, with no difference between 0% control groups. These results help to elucidate the etiology of subfailure injury and rupture in functionally distinct tendons.