Extended-spectrum beta-lactamases (ESBLs) are enzymes that hydrolyze the β-lactam ring, and ESBL-producing Escherichia coli is the most common cause of different human and animal infections. The aim of this study was to characterize ESBL-producing E.coli isolates genotypically in order to elucidate the presence of virulence isolates in aquaculture regions. A total of 40 ESBL-producing E. coli which were isolated from farmed fish (striped catfish, red tilapia) and wild fish (unidentified species) collected from Vinh Long, Dong Thap, and An Giang provinces. The results significantly indicated that of the 40 ESBL producers, 6 (15%) possessed CTX-M-1genes, 7 (17.5%) possessed CTX-M-9 and 6 (15%) had TEM genes. The SHV gene was not present among the isolates. However, 21 (52.5%) of the isolates had both CTX-M and TEM genes. In total, 17 (42.5%), 17 (42.5%), 3 (7.5%), 3 (7.5%) of ESBL-producing E.coli isolates clustered in group A, B1, B2, and D, respectively. Our study serves as a scientific basis for further research about the source of infection, characteristic transmission, and pathogenicity of ESBL-producing E.coli in the Mekong Delta.