Achilles tendon ruptures are traumatic injuries, and techniques for assessing repair outcomes rely on patient-based measures of pain and function, which do not directly assess tendon healing. Consequently, there is a need for a quantitative, in vivo measure of tendon properties. Therefore, the purpose of this study was to validate ultrasound imaging for evaluating collagen organization in tendons. In this study, we compared our novel, high-frequency ultrasound (HFUS) imaging and analysis method to a standard measure of collagen organization, crossed polarizer (CP) imaging. Eighteen mouse Achilles tendons were harvested and placed into a testing fixture where HFUS and CP imaging could be performed simultaneously in a controlled loading environment. Two experiments were conducted: (1) effect of loading on collagen alignment and (2) effect of an excisional injury on collagen alignment. As expected, it was found that both the HFUS and CP methods could reliably detect an increase in alignment with increasing load, as well as a decrease in alignment with injury. This HFUS method demonstrates that structural measures of collagen organization in tendon can be determined through ultrasound imaging. This experiment also provides a mechanistic evaluation of tissue structure that could potentially be used to develop a targeted approach to aid in rehabilitation or monitor return to activity after tendon injury.