Betanodaviruses are causative agents of neurological disorders in several species of fish. We cloned and sequenced the RNA2 segment of two grouper viruses isolated from Epinephelus malabaricus (malabaricus grouper nervous necrosis virus, MGNNV) and Epinephelus lanceolatus (dragon grouper nervous necrosis virus, DGNNV). The sequences of the two RNAs were 99% identical and comparison with previously sequenced RNA2 segments of fish nodaviruses striped jack nervous necrosis virus, Atlantic halibut virus, sea bass encephalitis virus, and greasy grouper nervous necrosis virus (GGNNV) revealed that MGNNV and DGNNV were most closely related to GGNNV. No correlation of sequence with geographical habitat was detected. The MGNNV coat protein, the gene product of RNA2, was expressed in Sf21 cells with a recombinant baculovirus system and virus-like particles (VLPs) spontaneously formed. Two types of VLPs were observed: a slower sedimenting particle was RNase-sensitive and stain-permeable, while the faster sedimenting particle survived RNase treatment and was not stain-permeable. An image reconstruction of the latter, obtained with electron cryomicroscopy data, revealed a morphology consistent with T = 3 quasi-symmetry but with features significantly different from insect nodavirus structures at the same resolution. This assembly system allows the first biophysical comparisons of fish and insect nodavirus structure, assembly, and stability.