Objective- To evaluate the feasibility and repeatability of in vivo measurement of stiffness gradients by means of acoustoelastography in the superficial digital flexor tendons (SDFTs) of clinically normal horses. Animals- 15 clinically normal horses. Procedures- For each horse, stiffness gradient index and dispersion values for SDFTs in both forelimbs were evaluated in longitudinal orientation by use of acoustoelastography at 3 sites (5, 10, and 15 cm distal to the accessory carpal bone) by 2 observers; for each observer, data were acquired twice per site. The left forelimb was always scanned before the right forelimb. Lifting of the contralateral forelimb with the carpus flexed during image acquisition resulted in the required SDFT deformation in the evaluated limb. Interobserver repeatability, intraobserver repeatability, and right-to-left limb symmetry for stiffness gradient index and dispersion values were evaluated. Results- Stiffness gradient index and dispersion values for SDFTs at different locations as well as effects of age or sex did not differ significantly among the 15 horses. Interclass correlation coefficients for interobserver repeatability, intraobserver repeatability, and limb symmetry revealed good to excellent agreement (intraclass correlation coefficients, > 0.74). Conclusions and Clinical Relevance- Results indicated that acoustoelastography is a feasible and repeatable technique for measuring stiffness gradients in SDFTs in clinically normal horses, and could potentially be used to compare healthy and diseased tendon states.