Infectious spleen and kidney necrosis virus (ISKNV) was first detected in imported ornamental fish in Korea in 2004.Herein, ISKNV was re-detected in ornamental fish imported to Korea from Singapore.Diseased dwarf gourami (Trichogaster lalius) imported from Singapore exhibited typical clin. signs associated with ISKNV infection, including darkened body coloration and splenomegaly.Through conventional PCR, ISKNV positivity was identified in 26.7% (4/15) of the cases, which differed from the 60% positivity (9/15) determined by real-time PCR.Furthermore, histopathol. lesions exhibited basophilic dysplastic hypertrophied cells and melano-macrophage center (MMC) formation in the spleen and kidney tissues, indicating chronic manifestations of ISKNV infection.Thus, it appears that the dwarf gourami suspected of being infected with ISKNV were imported from Singapore.Furthermore, homogenate-inoculated dwarf gourami fin (DGF) cells (a mixture of spleen and kidney tissues) displayed the typical cytopathic effects of ISKNV infection, including enlarged and rounded cells, and the newly identified ISKNV (DGIV-SAY-23) was successfully isolated.Moreover, virions with icosahedral capsid symmetry, a general morphol. feature of the genus Megalocytivirus, were identified using transmission electron microscopy.Based on the phylogeny of the major capsid protein (MCP) and ATPase (ATPase) genes, DGIV-SAY-23 belongs to ISKNV subtype I.The MCP gene showed 99.85% identity with DGA 10/8 isolated in Singapore in 2000.Mortality was observed in artificially infected ornamental fish for 14 days.The cumulative mortality rate of artificially infected pearl gourami (Trichogaster leeri) was 100%.The recurrent detection of ISKNV in imported ornamental fish in Korea supports the need for enhanced quarantine measures to prevent the spread of infectious diseases through international trade.To achieve an enhanced quarantine system, an appropriate detention period for live ornamental fish should be applied in the isolation facility based on the disease incubation period.