The prevalence of multiple emerging diseases including acute hepatopancreatic necrosis disease (AHPND) has caused serious economic losses of crustacean aquaculture industry in China in last several years. In order to analyzing the possible pathogenic agents related to the slow growth of Portunus trituberculatus from Weifang City, Shandong Province, the presence and infection of nine suspected pathogens in the disease P. trituberculatus individuals were investigated by using the molecular detection methods in present study. The nine pathogens included white spot syndrome virus (WSSV), Infectious Hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), AHPND-causing Vibrio parahaemolyticus (Vp_AHPND), enterocytozoon hepatopenaei (EHP), covert mortality nodavirus (CMNV), yellow head virus (YHV) and hepatopancreatic parvovirus (HPV). Meanwhile, histopathological analysis and in situ hybridization (ISH) were performed on the clinical samples. Results of molecular analysis showed that the P. trituberculatus samples were determined to be positive of Vp_AHPND and negative of other seven pathogens. Multiple sequence alignment based on the amplicons of Vp_AHPND nested-PCR of the samples revealed that sequences of the amplicons from P. trituberculatus samples shared 99% similarity to the pirA^vp virulence gene from the pathogenic V. parahaemolyticus. Phylogenetic analysis indicated that the sequence from P. trituberculatus samples was clustered into the same branch with the reported PirA of pathogenic V. parahaemolyticus. Histological examination revealed necrosis of epithelial cells in hepatopancreas tubules, myonecrosis of cardiac muscle in heart, necrosis of epithelial columnar cells of gills, severe damage of nerve cell in the thoracic ganglion, as well as nuclear pyknosis in these tissues. Micrographs of ISH showed that blue-violet hybridization signals of the Vp_AHPND probes were present in the lesions of the hepatopancreas, myocardium, gills, and thoracic ganglia. The results indicated that the P. trituberculatus samples from the shrimp and crab polyculture ponds were naturally infected by Vp_AHPND, which caused serious pathological damages of the hepatopancreas, myocardium, gills, and thoracic ganglia. The present study revealed, for the first time, that Vp_AHPND infection in P. trituberculatus and the pathological changes caused by the infection. The results provided basic information for revealing the natural host species of Vp_AHPND, and for the prevention and control of AHPND of P. trituberculatus.