In October 2022, an infectious disease outbreak was observed in farmed Portunus trituberculatus in Zhoushan, China. Samples of diseased crabs showing unspecific signs, such as anorexia, slow activity, and gill edema, were analyzed using parasitology, microbiology, histopathology, electron microscopy, and molecular identification. Parasitological and microbiological assays indicated that the diseased crabs were not infected with parasites or bacteria. The crab samples were tested by PCR methods recommended by World Organization for Animal Health and demonstrated to be free of Hematodinium, white spot syndrome virus (WSSV), mud crab discistrovirus-1 (MCDV-1), and Scylla serrata reovirus (SSRV). Histopathological examination revealed eosinophilic inclusions in hematopoietic tissue and hemocytes in gills, hepatopancreas, and the myocardial tissue of diseased crabs. Meanwhile, transmission electron microscopy showed that the virus in diseased crab tissues exhibited a typical icosahedral structure with a mean diameter of 150 nm, which was similar to the morphological characteristics of Decapoda iridescent virus 1 (DIV1). Nested PCR detection of DIV1 result showed that the first step of the PCR produced a 457 bp amplicon and the second step of the PCR produced a 129 bp amplicon. Phylogenetic analyses using gene sequences of major capsid protein (MCP) and ATPase revealed amplified sequences, DS.MCP201010 and DS.ATP202210 sequences, had the highest homology with the MCP gene and ATPase gene of DIV1, 100%. Therefore, it was determined that the pathogen of the disease was DIV1. In addition, specific probes directed to the MCP and ATPase gene of DIV1 were designed to obtain the distribution of virus particles in the hepatopancreas, heart, muscles, and gills of diseased crabs by fluorescence in situ hybridization, which was consistent with the results of electron microscopy and nested PCR. This study presents the first report of DIV1 infection in cultured Portunus trituberculatus, which will provide a strong reference for the prevention and control of the disease.