This study examined the genomes of A. hydrophila and A. dhakensis isolated in catfish farms and human patients in the Mekong Delta. Comparing with ANI results, it was found that rpoD sequencing had high discriminatory power between the two species, while aerA PCR and MALDI-TOF methods were not able to identify A. dhakensis. Also, A. dhakensis was more prevalent than A. hydrophila in both catfish and human isolates and MLST analyses revealed that ST251 A. hydrophila and ST656 A. dhakensis were the predominant sequence types, accounting for 69.2% and 52.7% of their respective species, while human-derived strains showed greater diversity. Both A. hydrophila and A. dhakensis exhibited multifactorial pathogenicity, but there were notable differences between the two species, including the incomplete or absent polar flagella and Type IV tap/msh/flp pili, as well as the absence of ast in all A. dhakensis isolates. Species-specific analysis revealed a higher diversity of virulence genes in ST656 A. dhakensis and ST251 A. hydrophila compared to other sequence types, while human isolates had fewer virulence factors compared to those from aquaculture sources, indicating host-specific pathogenesis. Both A. hydrophila and A. dhakensis strains displayed resistance to a wide range of antibiotics, with ST251 A. hydrophila possessing dfrA, sulI/II, arr-2, and tetA, and ST656 A. dhakensis exhibiting BlaAQU3, imiH, qnrS2, sulI/II, dfrA, floR, and tetA, but the level of multiple antibiotic-resistance was reduced after 2020. Overall, this study provides important insights into the characteristics of A. dhakensis and A. hydrophila in the Mekong Delta, serving as a foundation for future research on the prevention of MAS-causing Aeromonas.