Dorsal lacrimal glands, superior glands of the third eyelid and Harderian glands (deep gland of the third eyelid) from 19 bison and 18 cattle free of apparent ocular disease were examined to compare the normal anatomical properties of these glands. All glands were characterized and measured (length and width). The gross anatomy of the dorsal lacrimal glands was similar, with the exception of a bipartite gland in cattle. The bison's superior gland of the third eyelid and Harderian gland was longer as compared with cattle. A subset of the bison and cattle samples (five bison and five cattle) was sectioned for histological and histochemical analysis. The histology of the dorsal lacrimal and superior gland of the third eyelid revealed tubuloalveolar cells with basophilic vacuolated cytoplasm in bison and eosinophilic granular cytoplasm in cattle. The Harderian glands consisted of a tubuloalveolar anterior part combined with large lumens acini lined with cuboidal epithelium in the posterior part; the posterior part of the bison Harderian gland was more predominant than in cattle samples. Mucosubstance histochemistry revealed acidic and neutral glycoproteins with similar staining patterns in all glands of both species.