Abstract: In October 2020, some Pelodiscus sinensis died in a farm in Hubei Province, China. To determine the pathogen and pathogenic characteristics of the disease, a dominant strain A3 was isolated from the liver of the diseased P. sinensis. Based on physiological and biochemical characteristics and 16S rRNA gene sequence identification, it was identified as Citrobacter sp.. Further identification of the recN gene showed that the recN gene sequence identities between A3 and C. braakii were 95.58%-96.01%, while the identities between A3 and other species of Citrobacter were less than 93.72%, which indicated that A3 belonged to a novel species of Citrobacter. Artificial infection tests confirmed its pathogenicity to P. sinensis. Compared with Aeromonas hydrophila, the course of A3 infection was slower, but the mortality was as high. Drug sensitivity tests showed that strain A3 was sensitive to 10 kinds of antibiotics such as meropenem, and resistant to 9 kinds of antibiotics such as florfenicol. Multilocus sequence typing (MLST) showed that strain A3 belonged to a new sequence type (ST). A phylogenetic tree based on the 7 housekeeping gene sequences of Citrobacter from MLST database was constructed. The results showed that the evolution of Citrobacter spp. had no significant relationship with the source and location of isolates. Moreover, strain A3 has the closest genetic relationship with ST120 strain Citfre2580 isolated from human in Europe. In this study, the pathogen of P. sinensis was isolated and identified, and a more reliable method for the identification of Citrobacter was described, in order to arouse people's attention to the pathogenicity of Citrobacter, and provide some reference for the healthy aquaculture of aquatic products.