Tilapia (Oreochromis sp.) is the most important cultured fish in the 21 century and Indonesia is the country's second largest tilapia producer after China. It make Indonesia should be more concerned with any possible attack diseases that can infect both from the class of parasites, fungi, bacteria, and viruses especially Virus Nervous Necrosis (VNN). The existence of VNN in tilapia is one of the problems that must be handled. In the present research, a natural outbreak of VNN in tilapia (Oreochromis sp.) farmed in mid-Java in Indonesia was investigated using molecular genetic characterization. Research conducted using Reverse transcriptase PCR (RT-PCR) methods, sequencing analysis, and phylogenetic analysis. the construction of phylogenetic trees is based on the neighbor-joining method using software MEGA6. The samples used are a tilapia juvenile size 5-7 cm, with or no clinical symptoms, and taken at random in the cultivation of tilapia in Central Java. It showed that the tilapia juvenile infected VNN show changes and damage to the organs and tissues. It can be seen from the examination of clinical symptoms, anatomic pathology and histopathology. DNA of the tilapia infected by VNN was 294 bp on both the brain and the eyes organ. Phylogenetic analysis results showed that VNN from infected tilapia was the same as snapper and grouper and has the closest kinship with genotype Redspotted grouper nervous necrosis virus (RGNNV), which is about 99%. It can be stated that Betanodavirus in freshwater fish potential is derived from Betanodavirus in seawater fish.