The occurrence of mass mortality of Hybrid Groupers (Epinephelus sp.) in City of Grouper, Situbondo Regency hurts local and national grouper production which has resulted in the decline of grouper production. During the period February 2018 - April 2018 there was high mortality in Hybrid Grouper cultivation. This study identified the cause of mortality Hybrid Grouper. Fish samples obtained from 8 hatcheries in the City of Grouper area; each hatchery was taken as a sample of healthy-looking fish, sick symptoms of fish, and freshly death fish and samples were observed in clinical symptoms, histology, and RT-PCR to confirm the pathogen causing death Hybrid Grouper. The results of the analysis show that the body colour is dark, the tail fin starts to wriggle, swim on the surface of the water in reverse, weak movements, appetite decreases drastically, and the stomach looks enlarged, histological analysis shows vacuolation in the eye and brain organs, and analysis nested RT-PCR also shows the visible band 294 bp. This study shows that the cause of mass mortality Hybrid Groupers in City of Grouper was caused by attacks of viral nervous necrosis (VNN).