In 1995 and again in 1998, millions of pilchards (Sardinops sagax neopilchardus) were found dead or dying off the coast of Australia and also in New Zealand. The epizootics moved progressively, at a rapid speed against the prevailing currents. A previously unrecognised herpesvirus, Pilchard herpesvirus (PHV), was identified as the causative agent. Until recently, rapid and sensitive methods to detect PHV were not available and based on a previously identified and conserved 373 bp region of the genome, polymerase chain reaction (PCR), in situ hybridisation and real-time PCR methods were developed for the specific detection of PHV in formaldehyde-fixed and frozen tissues of pilchards. Real-time PCR was shown to have greater sensitivity than a conventional PCR and in situ hybridisation for the detection of PHV infection. The PCR assay and sequence analysis of the amplification products was used to compare the 373 bp region of the genome from strains obtained during the 1995 and 1998 epidemics. Significant differences between the strains were not detected. Additional sequence data was obtained adjacent to the 373 bp of known PHV sequence, which did not match any sequence in any of the genetic databases, and this will be invaluable for further study of the pilchard herpesvirus and the development of improved detection methods. The molecular-based methods of virus detection developed were applied to a reexamination of virus in paraffin-embedded tissues taken from fish during an attempt to transmit the virus to wild caught pilchards in 1999. The results obtained confirmed previously equivocal results that transmission of PHV to wild caught pilchards was achieved, although this experiment failed to demonstrate that transmission of the virus resulted in severe lesions typical of those seen in the epizootics. Using formaldehyde-fixed samples from fish collected during the 1998 PHV epizootic, virus was detected in fish collected 4 days prior to the occurrence of the epizootic even though the fish then appeared clinically normal, during the epizootic, and 8 days after mortalities had ceased. An investigation of wild pilchards collected from 4 Australian pilchard subpopulations using real-time PCR demonstrated that PHV was present in the gills of 13.75% of 800 fish sampled, indicating that the virus is now endemic in the Australian pilchard population. Variation in the prevalence of PHV infection in the 4 subpopulations was detected, higher in western and southern populations than in populations from the east coast. The endemic nature of PHV infection in the pilchard population explains why there have been no further epizootics with mass mortalities since 1998.