Pseudomonas aeruginosa uses Type III Secretion System (T3SS) to inject four types of secretion virulence determinants directly into the cytoplasm of host cell. This study aimed to determine the prevalence of virulence genes encoding type III secretion system toxins among P. aeruginosa isolates. A total of 51 isolates of P. aeruginosa were collected from different clinical samples in 2015-2016. Detection of gene sequences encoding type III secretion toxins ExoS, ExoT, ExoU and ExoY was performed by the multiplex PCR. Thirty-three of these P. aeruginosa isolates were genotyped by RAPD-PCR and Antibiogram for all isolates was also determined. Results of this research showed that the frequency of gene sequences encoding for type III secretion toxins detected by PCR in tested P. aeruginosa isolates was 100% and 72.5% for exoT and exoY, respectively, while exoS and exoU genes were not detected in these isolates. RAPD-PCR analyses showed that the tested isolates had 3 identical clones recovered from different hospitals and different geographical areas. The isolates of P. aeruginosa showed high resistance against Trimethoprim/Sulfamethoxazole (100%), Nalidixic acid (98%), Ceftriaxone (96.1%), Cefotaxim (96.1%) and Tetracycline (74.5%). To our knowledge, up to now, this is the first study documented the virulence determinants associated with P. aeruginosa in Palestine. Although our study is comprised of a relatively small number of P. aeruginosa isolates, it is a representative sample giving a picture of the general situation in Palestine. In conclusion, the results of this study showed high prevalence of T3SS genes among clinical isolates of P. aeruginosa in Palestine.