Background: Pseudomonas aeruginosa (P.aeruginosa) is one of the most clinically important bacteria. It can cause serious infections because it has many virulence factors which are the main reasons for emergence of antibiotic resistance. The most important one is type III secretion system (T3SS) that plays a main role in bacterial invasion and poor clinical outcome. Objectives: This study aimed to detect some phenotypic virulence factors and the antimicrobial susceptibility patterns among clinical isolates of P. aeruginosa, and to evaluate the prevalence of T3SS virulence genes among these isolates. Methods: The study included one hundred strains of P.aeruginosa isolated from different clinical specimens from patients in Menoufia University Hospitals. The isolates were identified by microbiological methods and tested for biofilm formation, bile esculin hydrolysis, hemolysin and DNase production. Antimicrobial susceptibility of the isolates was determined by disc diffusion method. Multiplex PCR technique was used for targeting the virulence genes. Results: virulence factors were recorded as follows: hemolysin (72%), biofilm formation (77%) and bile esculin hydrolysis (40%). The most frequent genes were exoT and exoY genes (90% and 77%) respectively, while the least detected one was exoU (27%).  High antibiotic resistance was detected to most of the used antibiotics. The exoT, exoY and exoS genes were significantly associated with high level of antibiotic resistance (p < /em><0.001). Conclusions: This study highlights the prevalence of T3SS virulence genes among clinical isolates of P.aeruginosa obtained fom different clinical specimens, Moreover, there was association between exoT, exoY and exoS genes and antimicrobial resistance in these isolates.