Objective:To investigate the distribution of herpesvirus DNA in the corneal layers of herpesvirus-positive corneal transplantation patients and to compare the efficiency of viral DNA detection in corneal and aqueous humor samples in these patients.Methods:A diagnostic test study was conducted.Data from patients, who underwent keratoplasty in Peking University Third Hospital from May 2015 to August 2021 and tested positive for herpesvirus in corneal tissue and/or aqueous humor samples obtained during surgery, were collected through the clinical medical record system.The demographic data and virus distribution layers of these patients were analyzed.The rate of herpesvirus detection in different samples was analyzed.The sensitivity of different samples for viral DNA detection was analyzed by receiver operating characteristic curves, and area under the curve (AUC) and 95% confidence interval ( CI) were recorded.This study adhered to the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of Peking University Third Hospital (No.M2021283).Written informed consent was obtained from each patient before entering the cohort. Results:A total of 166 herpesvirus-positive patients (166 eyes) were collected.Of the 166 eyes, 75 eyes (45.2%) were positive for cytomegalovirus (CMV), 34 eyes (20.5%) for herpes simplex virus 1 (HSV-1), 30 eyes (18.1%) for varicella zoster virus (VZV), and 27 eyes (16.3%) for Epstein-Barr virus (EBV).CMV DNA and VZV DNA were detected in the endothelial layers of 47 eyes (62.7%) and 26 eyes (86.7%), respectively, which were significantly higher than the 28 eyes (37.3%) and 4 eyes (13.3%) with virus located in stromal layers ( χ2=4.813, 16.133; both at P<0.05).HSV-1 DNA and EBV DNA were detected in the endothelial layer of 8 eyes (23.5%) and 5 eyes (18.5%), respectively, which were less than the 26 eyes (76.5%) and 22 eyes (81.5%) with virus located in stromal layers, and the differences were statistically significant ( χ2=9.529, 10.704; both at P<0.001).The sensitivity of corneal samples for herpesvirus DNA positivity was 71.6%, which was higher than 54.1% of aqueous humor.The detection sensitivities of corneal samples for CMV DNA and VZV DNA positivity were 64.3%(AUC=0.821, 95% CI: 0.705-0.938) and 35.7%(AUC=0.679, 95% CI: 0.475-0.882), respectively, which were lower than 71.4%(AUC=0.875, 95% CI: 0.750-0.964) and 85.7%(AUC=0.929, 95% CI: 0.816-1.000) of aqueous humor samples.The detection sensitivities of corneal samples for HSV-1 DNA and EBV DNA were 100%(AUC=1.000, 95% CI: 1.000-1.000) and 92.3%(AUC=0.962, 95% CI: 0.875-1.000), respectively, which were higher than 27.8%(AUC=0.639, 95% CI: 0.455-0.823) and 23.1%(AUC=0.615, 95% CI: 0.395-0.835) of aqueous humor samples. Conclusions:The detection rate of CMV DNA is highest among herpesvirus-positive keratoplasty patients.CMV DNA and VZV DNA are primarily located in the corneal endothelial layers, while HSV-1 DNA and EBV DNA are more predominant in the corneal stromal layer.The sensitivity of virus DNA detection is higher in the cornea than in aqueous humor.