The virus in the genus Megalocytivirus, family Iridoviridae, has been considered one of the major pathogens causing mass mortalities in both wild and captive finfish in the Indo-Pacific region. In this study, disease investigation carried out between February and November 2018 focused on naturally diseased barramundi in inland growout farms located in Central/Eastern region of Thailand. According to PCR screening from liver and spleen, 60% of samples (20 out of 33) from 8 distinct outbreaks (30-90% cumulative mortalities) were infected by megalocytivirus. Viral taxonomy was subsequently identified by nucleotide sequencing of MCP and ATPase genes which indicated that infectious spleen and kidney necrosis virus (ISKNV) was the major megalocytivirus associated with die-off. In addition, scale drop disease virus (SDDV) was identified from one outbreak manifesting extensive scale loss lesion. Concurrent infection of megalocytivirus and pathogenic bacteria, e.g. Flavobacterium columnare, were often found (Table 1). Phylogenetic analysis revealed that all ISKNV isolates shared 100% sequence identity and was categorized as genotype I, based on MCP gene sequence. Cumulative mortality of 90% (n = 50) was observed in juvenile barramundi at 21 days after experimental challenge via i.p. injection with GF cell propagated ISKNV. Affected fish exhibited ascites, darkened body and hemorrhage, while necrosis, inflammation and nuclear pyknosis/karyorhexis was observed histopathologically. Lastly, high-throughput sequencing of naturally infected specimen was able to recover the complete genome of ISKNV (111.4 – 111.6 kb in length, Fig. 1) which was almost identical to ISKNV reference strain (0.02% genetic difference).