Congenital corneal opacity (CCO) is a rare disorder. Penetrating keratoplasty (PK) is the main surgical option for CCO, but many factors affect graft survival. Therefore, this study aimed to perform a virological examination of CCO specimens after PK to explore the relationship between virological factors and graft survival after PK.This prospective study included consecutive patients (<6 months of age) diagnosed with CCO and treated with PK at Beijing Tongren Hospital from August 2017 to January 2018. Next-generation sequencing was used to detect viral DNA in the CCO specimens. The survival of the primary graft was analysed using the Kaplan-Meier method.Overall, 24 eyes of 24 infants were treated with PK during the study period. The mean age at surgery was 4.8±1.1 months. Epstein-Barr virus DNA was detected in two specimens, varicella-zoster virus DNA in one specimen, herpes simplex virus DNA in three specimens and cytomegalovirus DNA in one specimen. In the virus-positive group, only one (14.3%) graft remained clear during follow-up. In contrast, in the virus-negative group (n=17), 13 (76.5%) grafts were still clear at the last follow-up. The mean survival of the grafts in the virus-positive group was significantly shorter than in the virus-negative group (11.0±9.8 months vs 27.1±7.7, p<0.001).The presence of viral DNA in CCO specimens might be associated with poor graft survival after PK.© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.