Red sea bream iridovirus (RSIV), a member of the genus Megalocytivirus within the family Iridoviridae, is one of the most economically important viral pathogens affecting marine aquaculture in Asia. Although RSIV has been extensively reported in Japan, South Korea, China, Taiwan, and several Southeast Asian countries, information regarding its occurrence in Vietnam remains limited. In the present study, RSIV infection was investigated in cultured Asian seabass (Lates calcarifer) collected from marine cage farms in coastal provinces of Vietnam. Diseased fish exhibited typical clinical signs including lethargy, anorexia, body darkening, abdominal distension, anemia, gill hemorrhage, and splenomegaly. Approximately 40% of sampled fish tested positive for RSIV by PCR targeting the major capsid protein (MCP) gene. The virus was successfully isolated and propagated in GF cell lines, where cytopathic effects appeared 4–5 days post inoculation and were characterized by enlarged rounded cells followed by cell detachment. MCP gene sequencing revealed extremely high nucleotide similarity (99.85%) with RSIV isolate KagYT-96 from Japan. Phylogenetic analysis clustered the Vietnamese isolate together with RSIV type I strains from Japan, China, South Korea, and Taiwan. Serial passages of the virus in GF cells demonstrated high genetic stability of the MCP gene through at least 15 passages. Experimental challenge of healthy seabass with the isolated strain KH63 resulted in 100% cumulative mortality within 10 days post infection, confirming the high virulence of the isolate. To our knowledge, this study represents the first molecular confirmation, isolation, and characterization of RSIV infection in cultured Asian seabass in Vietnam. These findings highlight the urgent need for enhanced surveillance, biosecurity management, and preventive strategies against RSIV in Vietnamese marine aquaculture.