Ornamental fishes were collected from 173 locations in nine provinces of Thailand during 2016-2018. Using real-time PCR analysis, twelve fish species of three families (Poeciliidae, Cichlidae and Osphronemidae) were found positive for Megalocytivirus, a pathogenic agent which causes a significantly high mortality in a wide range of aquatic animal hosts. A geographic information system (GIS) displayed the distribution of megalocytivirus to the northern, central and western regions of Thailand. Nevertheless, one sampling province showed megalocytivirus-free status and the overall rate of virus occurrence declined by 10% during the study period. The transport and translocation of ornamental fish could be a key route for megalocytivirus introduction to new geographical areas. Molecular phylogenetic analysis based on the major capsid protein (MCP) confirmed that Megalocytivius in the current study belongs to members of an infectious spleen and kidney necrosis virus (ISKNV) cluster. Although most infected fish were asymptomatic, non-specific symptoms such as haemorrhages, loss of coloration on the fish body and pale liver were observed without substantial mortality. Cytomegalic cells with inclusion bodies were examined. Issues associated with host molecular defense mechanism, persistent infection, and mortality risk factors will await further study.