Abstract: Aeromonas hydrophila is a Gram-negative bacterium, which is widely distributed in the water environment. Pathogenic strains can cause sepsis in a variety of aquatic animals. In Sept. 2015, crucian carp (Carassius carassius) in one farm of Nanjing showed symptoms of haemorrhagic septicaemia. Samples from diseased fish in three ponds were collected and the pathogenic bacteria were isolated. Three A. hydrophila isolates were identified based on sequencing of the housekeeping gene gyrB, and named GL-15, GL-17 and GL-18, respectively. Then the three A. hydrophila strains were typed using multilocus sequence typing (MLST) based on six housekeeping genes. Also, all the three isolates were analyzed for hemolytic and proteolytic characterizations and pathogenicity in zebrafish. The results showed that all the three strains presented hemolytic and proteolytic activities, and the abilities of biofilm formation in which OD₅₉₅ is more than 0.5 and also had swarming and swimming motility. And they were highly virulent strains, with the LD₅₀ values of 1.4×10³ to 3.9×10³ CFU. This study provided a basis for control of MAS caused by A. hydrophila in Nanjing area.