In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen over their entire geographical range on the Australian coastline. A virus with typical herpesvirus morphology was identified as the causative agent, although the source of the virus remains unknown. At the time of the mortality events, the only available diagnostic test available for the detection of the virus was electron microscopy and hence, development of a rapid diagnostic test for detection and identification of the virus was required. Initial sequence data for Pilchard herpesvirus (PHV) was acquired by comparing the highly conserved region of the ORF 62 gene of other piscine herpesviruses (Ictalurid herpesvirus 1 [Channel catfish virus, CCV] and Salmonid herpesvirus 2 [Oncorhynchus masou virus, SaHV2, OMV]), and designing primers that successfully amplified a fragment of PHV. Here we describe the use of polymerase chain reaction (PCR) technology to detect PHV in infected tissues. Sequence analysis of amplified fragment resulting from this PCR is different from all known herpesviruses and can therefore distinguish PHV from all known viruses.