The morphology of a duplication phenomenon of the canine Descemet's membrane (DM) is described in relation to signalment, history, and ocular disease status. Sixty-six canine eyes from the Comparative Ocular Pathology Laboratory of Wisconsin archives between 2000 and 2007 were examined. All cases were stained with hematoxylin and eosin and Alcian blue periodic acid-Schiff (PAS), while 14 cases were additionally stained with Masson's trichrome, picrosirius red, cytokeratin AE1/AE3 (CK), vimentin, and alpha-smooth muscle actin (SMA). Transmission electron microscopy (TEM) examination was performed in 3 corneas and in 1 normal control eye. Alcian blue PAS staining and TEM confirmed the basement membrane nature of the abnormal secondary DM. The thickness of the first DM, referred to as the corneal layer (CL) and the second or anterior chamber layer (ACL), were nearly the same, with no significant difference seen (P = .93). In 39% (26/66) of the eyes, a fibrous, collagenous matrix component was present between the CL and ACL, which contains vimentin-positive and alpha-SMA-negative spindle cells (14/14).The corneal endothelial cells in 7/14 eyes stained weakly with CK and strongly in 2 additional eyes. The most frequent histopathologically confirmed, clinical ocular histories were chronic glaucoma in 76% (50/66) of eyes, previous intraocular surgery in 36% (24/66), lens luxation in 21% (4/66), and blunt trauma in 15% (10/66) of the cases. We speculate that activation and migration of endothelial cells, in association with trauma or lens contact, play a role in the pathogenesis of this phenomenon.