• PURPOSE: To evaluate the use of impression cytology in the diagnosis of Acanthamoeba keratitis with surface involvement. • DESIGN: Observational case series. • METHODS: Three patients with culture-proven superficial Acanthamoeba keratitis were studied. Patients' medical records were reviewed for clinical information, treatment procedures and disease course. Impression cytology specimens taken from the cornea of those patients were processed and observed by bright field microscopy. The main outcome measure was the cytopathological examination and identification of amoebic organisms. • RESULTS: In each of these three patients, impression cytology specimens revealed the amoeba cysts. Some specimens also showed pleomorphic trophozoites with typical amoeba nuclei. • CONCLUSION: Corneal impression cytology successfully detected the cysts and trophozoites of Acanthamoeba from patients with superficial amoeba keratitis. This indicates that impression cytology can be a useful technique to facilitate early diagnosis of Acanthamoeba keratitis with surface involvement.