The emergence of diseases has caused much health and economic damage. Viral Nervous Necrosis (VNN) is considered as one of the most important threats to aquatic ecosystems. VNN can cause severe mortality and economic loss in fish farms. The high water temperatures in southern Iran and the observed incidences of fish mortality in the Persian Gulf led to the hypothesis of the possible emergence of VNN. Therefore, this study aimed to monitor two species of fish susceptible to VNN using PCR, and Nested PCR methods and comparing the sensitivity of these methods to the identification of Betanodavirus infection in apparently healthy and symptomatic fish. About 850 Grouper (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) fish of the Persian Gulf were collected randomly and examined. Molecular methods were used to identify NNV in visibly healthy and symptomatic fish of the Persian Gulf of Iran. The results of the PCR showed no positive cases, but the Nested PCR revealed some positive results. Then, the phylogenetic analysis of the virus sequence was performed. The nucleotide sequence of Nested PCR products revealed a 98-100% homology with Red Spotted Grouper Viral Nervous Necrosis (RGNNV). This is the first report on VNN tracing and detection as well as phylogenetic analysis of the virus from the Persian Gulf of Iran. Therefore, considering the importance of emerging viral diseases and the irreparable damage they cause, continuous monitoring and epidemiological studies of VNN were recommended by authorized organizations.