Background: Pseudomonas aeruginosa is the second most common pathogenic bacteria isolated from patients and a major cause of nosocomial infections. It can also cause both acute and chronic infectious illnesses. Being a virulent organism, P. aeruginosa has a propensity to acquire resistance to the majority of antibiotics used in therapy. Objective: This study aimed to detect certain virulence genes in P. aeruginosa isolates from various clinical sources. Patients and methods: A total of (200) clinical specimens were taken from the patients with various age groups (3-60) years, from (Al Yarmouk Teaching Hospitals) in Baghdad from beginning of December 2021 to the end of March 2022, different specimens included wounds, burns, ear and diabetic foot swabs.  All specimens were cultured on (MacConkey agar and cetrimide agar). On the other hand, bacterial virulence factors have been studied and they included detection of biofilm production using microtiter plate method (MTP), which is considered most sensitive. Detection of some virulence genes was performed using specific primers for each gene (PilB and LasB). Results: The culture results revealed 53 isolates belong to the Pseudomonas bacteria. Culture characteristic and Vitek2 system showed that all 53 isolates belonged to P. aeruginosa. The results showed 100 % from 53 isolated were adhesion and biofilm formation–positive by (MTP) on the OD 590 nm. The results showed that LasB gene was found in all bacterial isolates (100%) and PilB gene was detected in 56.6%. Conclusions:Molecular analysis showed that PilB and LasB were associated with formation of biofilm.