[en] Koi herpesvirus (KHV), recently designated in the species Cyprinid Herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp using bioluminescence imaging. Taking profit of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between ORF 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid: the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain encoding a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 hours post-infection; while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different time post-infection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish, covering the fins and also the body, is the major portal of entry of KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system nicknamed "U-tube" to perform per-cutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin and not the gills is the major portal of entry of KHV in carp.