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Background: Herpes simplex virus 1 (HSV-1) is a major cause of unilateral blindness worldwide. Diagnosis of disease is based on clinical examination. In this study, the prevalence of the virus in patients attending Feiz Ophthalmology Centre, Isfahan, Iran, was determined using polymerase chain reaction (PCR) method. Methods: This descriptive study was done during 2012-2013. 307 samples were taken from patients with clinical keratoconjunctivitis. DNA of HSV-1 was detected using PCR method. The data were evaluated using SPSS software. Findings: From 307 clinical samples, 182 (59.3%) were of women and 125 (40.7%) of men. 20 patients with the mean age of 27.55 ± 3.31 were positive for HSV-1. Conclusion: The prevalence of herpetic keratitis was 6.51%. The frequency is not related to sex.