Simple SummaryAlthough infection with herpesvirus in owls has commonly been described as a highly lethal disease, there is very little information about the presence of herpesvirus and its potential impact in living owls in wild populations. Our study detected herpesvirus in a breeding population of Ural owls, which showed no clinical signs of illness nor productivity deviances (i.e., in clutch and brood size). Herpesvirus was detected in Ural owl adults and chicks, but not in a young tawny owl (despite the fact that they were in same nest and in persistent contact). Furthermore, herpesviruses were also detected in yellow-necked mice as both owls’ main prey. However, comparison of the herpesviruses detected showed that different herpesviruses are present in the owls and mice. The results of this study show that herpesvirus may be present in a Ural owl breeding population without any consequences on health and breeding performance. However, in the case of tawny owls, it seems that they are not susceptible to infection, which could be related to their polymorphism. It seems that small rodents are not a source of herpesvirus infection in owls and that the probable herpesvirus transmission pathway takes place intraspecifically, mostly from adults to young.Birds are a frequent host of a large variety of herpesviruses, and infections in them may go unnoticed or may result in fatal disease. In wild breeding populations of owls, there is very limited information about the presence, impact, and potential transmission of herpesvirus. The herpesvirus partial DNA polymerase gene was detected using polymerase chain reaction in oropharyngeal swabs of 16 out of 170 owls examined that were captured in or near nest boxes. Herpesvirus was detected in Ural owls (Strix uralensis), in both adults and young, but not in tawny owls (Strix aluco). In yellow-necked mice (Apodemus flavicollis), as the main prey of tawny owls and Ural owls in the area, herpesvirus was detected in the organs of 2 out of 40 mice captured at the same locations as the owls. Phylogenetic analysis showed that the herpesvirus sequences detected in the Ural owls differed from the herpesvirus sequences detected in the yellow-necked mice. The results indicate that herpesvirus infection exists in the breeding wild Ural owl population. However, herpesvirus-infected owls did not show any clinical or productivity deviances and, based on a phylogenetic comparison of detected herpesvirus sequences and sequences obtained from Genbank database, it seems that mice and other rodents are not the source of owl infections. The most probable transmission pathway is intraspecific, especially from adults to their chicks, but the origin of herpesvirus in owls remains to be investigated.