<p>Tendon rupture is a traumatic injury that is difficult to recover to the condition before injury. In previous studies, histological staining and tensile testing have been widely used to evaluate histological and mechanical healing. However, since both methods are destructive and invasive, it is difficult to apply these methods to clinical diagnosis. If the degree of healing can be visualized nondestructively and noninvasively, new findings may be obtained regarding mechanisms of the tendon healing process. In this study, we used second-harmonic-generation (SHG) microscopy to evaluate the degree of healing of ruptured tendon in a rabbit model. SHG microscopy has high selectivity and high image contrast with respect to the structural maturity, density, and aggregates of collagen molecule, without the need for histological sectioning and staining. Furthermore, since SHG light intensity sensitively reflects the structural maturity of collagen molecule and its aggregates, it has the potential to be a good indicator for the degree of healing of the injured tendon. By comparing the SHG images between 4-week-healing tendons and normal tendons in the animal model, we confirmed that SHG light intensity of the healing tendon was significantly lower than that of the normal tendon, indicating that the collagen structure in the healing tendon is still immature. Furthermore, we performed image analysis based on 2D Fourier transform of the acquired SHG images, and confirmed a significant difference in collagen distribution depending on the sample. These results indicate that SHG microscopy has the unique potential as an indicator of tendon healing.</p>