Equine gammaherpesvirus 2 (EHV-2) has been linked to keratitis and keratoconjunctivitis but has also been isolated in horses showing no signs of disease. The aim of the current study was to assess the importance of EHV-2 infection in the aetiopathogenesis of ocular disease, where the applied treatment failed. Seventy-eight horses with nonhealing ocular disease were examined at the Equine Clinic of the University of Veterinary Medicine and Pharmaceutical Sciences, Brno, Czech Republic, between the years 2009 and 2016. In total, 96 conjunctival swabs were taken and, starting from 2014, peripheral blood leukocytes (PBLs) were also examined in 42 patients. Positive EHV-2 results were detected in 53 ocular swab samples (54.64%) and in 22 PBL samples (51.16%). The horses were divided into three groups according to age, up to 3 years, from 3 to 15 years and older than 15 years. Depending on the clinical presentation, horses were also divided into nonulcerative or ulcerative keratitis, keratouveitis, keratoconjunctivitis, and corneal degeneration groups. The group of young horses had a significantly higher ocular swab positivity compared to the middle group (P = 0.01). Increased bilateral ocular occurrence with decreasing age was observed, although it was not significant (P = 0.04). Significant correlation was confirmed between PBL samples and ocular swabs (P = 0.01). This correlation was even higher in cases of bilateral infection. No significant differences were detected when comparing the groups according to the clinical presentation. This study describes the prevalence of EHV-2 in different age group horses with non-healing keratopathies.