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Objective: To describe ultrasonography as a diagnostic method of in vivo Descemet's membrane detachment (DMD) in horses. Animals studied: Seven horses (three Icelandic horses, two Dutch Warmblood horses, one Appaloosa, and one Welsh Pony), presenting with moderate‐to‐severe focal or diffuse corneal edema, in whom DMD was suspected on ultrasonographic examination and confirmed with histopathology, were studied. Procedure: A retrospective analysis of case records of horses with suspected DMD was performed. Results: Median age at presentation was 14 years (range 11‐24). Clinical signs in eyes with DMD were unilateral in all horses and included blepharospasm and epiphora (6/7), buphthalmos (5/7), moderate‐to‐severe focal or diffuse corneal edema (7/7), corneal epithelial bullae (4/7), corneal neovascularization (4/7), Haab's striae (2/7), corneal endothelial precipitates (1/7), fibrin in the anterior chamber (1/7), focal cataract (2/7), and pigment deposits on the anterior lens capsule (1/7). During transpalpebral ultrasonography, a distinct linear echogenic structure was noted in the anterior chamber, initially diverging from, and later running parallel to, the posterior lining of the cornea in all eyes studied. In all cases, the cornea was severely thickened and echogenic, consistent with edema, and DMD was suspected. In all horses, the clinical signs progressed and the affected eye was eventually enucleated. Histopathology revealed DMD (7/7), spindle cell proliferation (4/7), Descemet's membrane reformation (3/7), and inflammation of the anterior uvea (5/7). Overall incidence was 1.04%. Conclusions: Ultrasonography is an adequate tool in diagnosing DMD in horses. Descemet's membrane detachment should be included in the differential diagnosis in horses with dense focal or diffuse corneal edema. [ABSTRACT FROM AUTHOR]