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This comparative study aims to assess the differences in the third eyelid gland and cartilage morphology and histology between dogs (Canis lupus familiaris) and cats (Felis catus). Twenty nictitans membranes were collected from 20 adult dogs and 20 adult cats. The nictitans membranes were dissected to examine the structure, shape, and positions of the nictitans gland and cartilage. Histologically, the samples were fixed in 10% neutral buffer formalin and then prepared for H&E stains. Generally, in both species, the cartilage consists of a long, slender body, and pigmented large dorsal and small ventral wings. The cartilage had a curving shape that resembled the eyeball's curvature and had two surfaces; palpebral and corneal or bulbar surface. At the basal end of the corneal surface, there are several lymph nodes aggregates. In the dogs, the nictitans cartilage was longer and slenderer than in the cats. The body of the cartilage was wrapped by a spherical and light pink nictitans gland in the dogs while the cats had oval nictitans gland. Microscopically, the nictitans gland in the dogs was composed of a compound tubuloacinar gland whereas; in cats, it was composed of a compound acinar gland. There was no difference histologically in the intralobular and the interlobar septa between species, the isogenous group of chondrocytes was higher in cats Vs. the dogs that showed few in number. In conclusion, the nictitans gland and cartilage were varied in their anatomical and histological structure. This should help researchers make preparations to prevent negative impacts and could help them investigate changes made more pronounced by disease conditions.