Viral nervous necrosis (VNN) is a worldwide disease among marine fishes. In Taiwan, NNN disease was first identified in 2 species of hatchery-reared grouper, Epinephelus fuscogutatus and E. akaaya in 1994. Since then, increasing mortalities have occurred among groupers Epinephelus spp., and also among European eels Anguilla anguilla L., yellow-wax pompano Trachinotus falcatus, firespot snapper Lutjanus erythropterus B., barramundi Lates calcarifer, cobias Rachycentron canadum, humpback groupers Cromileptes altivelis and Chinese catfish Parasilurus asotus. In the present study, samples were collected from affected fishes and processed for reverse transcriptase (RT) PCR amplification and virus isolation in cell culture. Infected cells (GF-1 cell line) exhibited cytopathic-effect characteristics of grouper nervous necrosis virus (GNNV). A RT-PCR product of approximately 830 bp was amplified from the brain homogenate of tested samples and sequenced. The nucleotide and deduced amino acid sequences of the amplified RT-PCR products from all isolates were strongly homologous (>97%) with the corresponding region of the published sequence of red-spotted grouper nervous necrosis virus (RGNVV). Therefore, all Taiwan NNV (nervous necrosis virus) isolates studied in this report belong to the RGNNV genotype. We used 5 neutralizing monoclonal antibodies (MAbs) against GNNV to analyze the antigenic relationship of Taiwan NNV isolates and striped jack nervous necrosis virus (SJNNV). The results of neutralization tests revealed that all Taiwan NNV isolates were closely related, but antigenically different from SJNNV in 3 neutralizing epitopes. To our knowledge, this is the first description of NNV infection in European eels, yellow-wax pompano, firespot snapper, cobia and Chinese catfish, and the first reported instance of natural NNV infection in freshwater fishes causing high mortality.