Abstract: In order to investigate the prevalence and classification of Aeromonas hydrophila from the freshwater fish in Zhenjiang City, A. hydrophila was purified from 74 strains of pathogenic bacteria isolated from several aquaculture farms in the city, and then the morphological observation, gyrB gene sequence analysis and multilocus sequence typing (MLST) were conducted. The results showed that A. hydrophila appeared as light yellow, round, smooth, and neatly edged colonies on RS agar plates and red, rod-shaped bacteria with blunt ends by microscopic examination after Gram staining. After conducting the sequence alignment analysis on the gyrB gene of isolated strains, 40 strains of Aeromonas were identified, of which 12 were preliminarily determined to be A. hydrophila. The phylogenetic tree of Aeromonas isolates based on the gyrB gene was further constructed, finally identifying eight isolates as A. hydrophila. The PCR amplification and sequence analysis were performed on the housekeeping genes gyrB, groL, gltA, metG, ppsA, and recA of A. hydrophila. Two strains of A. hydrophila type ST251, three strains of type ST378, and one strain each of types ST469, ST361, and ST1075 were identified. The results showed that there was a potential risk of the prevalence of ST251 type of highly pathogenic A. hydrophila in the region, which endangered the green and high-quality development of the local aquaculture industry.