Two recent case reports described a congenital keratinization defect (congenital follicular parakeratosis; CFP) in Rottweiler and Siberian Husky dogs. Skin biopsy specimens revealed marked parakeratosis targeting the hair follicle and numerous intracorneal vacuoles. A retrospective histopathological study was conducted on skin biopsy specimens from 111 dogs with diseases associated with parakeratotic hyperkeratosis to determine whether intracorneal vacuoles were present. Additional criteria evaluated were the size and location of the vacuoles and the degree of parakeratosis. Cases examined included dogs with primary idiopathic seborrhoea, necrolytic migratory erythema (NME), Malassezia dermatitis, zinc-responsive dermatosis, hereditary nasal hyperkeratosis of Labrador Retriever dogs, thallotoxicosis and CFP. Thirty-seven cases (37/111, 33%) had intracorneal vacuoles, including nine cases of primary idiopathic seborrhoea (9/29, 31%), 10 cases of NME (10/18, 56%), five cases of Malassezia dermatitis (5/19, 26%), five cases of zinc-responsive dermatosis (5/36, 14%), five cases of hereditary nasal hyperkeratosis (5/5, 100%) and three cases of CFP (3/3, 100%). If present, intracorneal vacuoles were found throughout all layers of the parakeratin. The sizes of intracorneal vacuoles varied among diseases, but large (> 5 microm) vacuoles only were present in CFP. Biopsies with a larger degree of parakeratosis were significantly more likely to have intracorneal vacuoles (P = < 0.001). Based on this study, intracorneal vacuoles are a common finding in many parakeratotic skin diseases of the dog, but large (> 5 microm) vacuoles are found only in CFP.