ABSTRACT: Diagnostic tests with high analytical sensitivity are required to detect Cyprinid herpesvirus-3 (CyHV-3) in carriers that have been implicated in the dissemination of this important disease of koi and common carp Cyprinus carpio. A nested polymerase chain reaction (PCR) test was developed to detect CyHV-3 DNA in tissues of infected fish. The 2-round PCR amplified 529 and 379 bp segments of the CyHV-3 gene coding for the major capsid protein using specific external and internal primers, respectively. The PCR test did not amplify genomic DNA from either Cyprinid herpesvirus-1 (CyHV-1) or Cyprinid herpesvirus-2 (CyHV-2). The nested PCR test detected CyHV-3 DNA at a 10-fold lower concentration than a routinely used, 1-round test. Hence, the new method should provide more sensitive diagnosis of CyHV-3 infection among koi and carp populations.