This study was conducted on 70 diseased Pangasius hypophthalmus fish collected from different Pangasius fish farms in Egypt. Bacteriological examination revealed the isolation of P. aeruginosa that was biochemically confirmed using MALDI-TOF MS and VITEK 2 compact system. The prevalence and virulence of Pseudomonas aeruginosa were estimated genotypically using species-specific 16S rDNA gene, OprL and ToxA virulent genes for detection of pathogenic strains followed by sequencing analysis. Pathogenicity of isolated Pseudomonas aeruginosa was confirmed by experimental intraperitoneal injection of healthy Pangasius hypophthalmus with positively virulent strains resulting in high mortalities among injected fish.