Grouper iridovirus in Taiwan (TGIV) is a major pathogen in marine fish aquaculture. Currently, diagnosis of TGIV infection relies mainly on histopathological examination. In this study, we present the first nested polymerase chain reaction (PCR) designed for early detection of TGIV. The detection limit of this method is 50 femtograms (fg; 1 fg = 10[super]-15 g) of target DNA extracted from infected fish DNA, 0.5 fg (100 copies) of plasmid DNA with the target viral insert, and 0.05 fg (10 copies) of plasmid DNA in the presence of 50 ng of fish DNA in a 25- mu L PCR setup. There was no amplification of DNA from healthy fish or from fish with lymphocystis disease. In contrast, the TGIV PCR resulted in the diagnostic amplicons of 1,339 base pairs (bp; round 1) and 305 bp (round 2) from both mildly and severely infected fish. Diagnostic amplicons were amplified from naturally infected hybrids of Malabar grouper Epinephelus malabaricus and red-spotted grouper E. akaara, giant seaperch Lates calcarifer, and largemouth bass Micropterus salmoides. The PCR detected TGIV infection in experimentally infected groupers at 1 d postchallenge, 2 d earlier than current histological examination. An in situ hybridization test using labeled primers from the TGIV PCR assay as probes detected virus-infected enlarged cells in hematopoietic tissues. The PCR procedure was proven to be a quick, reliable, and sensitive tool in TGIV detection. Several other primers designed for largemouth bass virus or red seabream Pagellus bogaraveo iridovirus were also tested. These PCR results and sequence analysis of the amplicons obtained by using the TGIV PCR assay suggest that the etiological agents infecting giant seaperch and largemouth bass might be the same and related to but distinct from TGIV.