Background: Pseudomonas aeruginosa is a pathogenic organism responsible for frequent wound and nosocomial infections worldwide. Its infections are difficult to control since the organism is known to rapidly develop antibiotic resistance and becomes multidrug-resistant (MDR) during treatment of patients. Aim of the study: This study was intended to investigate the occurrence of certain important types of (ESBL) and (MBL) enzymes in association with important specific virulence factors  associated with P. aeruginosa clinical isolates from Khartoum, Sudan. Methods: This study investigated 70 P. aeruginosa isolates which were collected from patients admitted to four major hospitals in Khartoum  (Fedail, Ribat, Ibn Sina and Soba hospitals). These isolates were recovered from 40 wound swabs (57.1%), 27 urine samples (38.6%), and 3 pleural fluid samples (4.3%) of patients. Higher numbers of isolates were recovered from males 42 (60%) than in females 28 (40%). All P. aeruginosa isolates were first confirmed by conventional biochemical and second using molecular PCR tests.   PCR methods were also used for detecting the presence of the virulence genes ToxA, AlgD, LasB, exoS, exoU, CTX, GES-1, and genes of VIM, IMP, KPC, CTX, VEB-1 and SHV-1. Results:   Antimicrobial susceptibility testing of P. aeruginosa isolates showed a high resistance to azetronam 49 (70%), followed by ceftazidime 32 (45.7%), 16 ciprofloxacin (22.9%), gentamicin 13 (18.6 %), piperacillin-tazobactam 11 (15.7%), amikacin 9 (12.9 %), and imipenem 6 (8.6%) showed the least resistance. All isolates were positive for algD and lasB (100%), followed by toxA (90%), exoS (34.3), exoU (24.3%), respectively. The rates of detected ESBL genes blaTEM, blaCTX-m, blaSHV-1,GES-1, were 3.3%, 6.6%, 10%, 3.3%,10%, respectively, but all isolates were negative for bla-KPC and bla- VIM and IMP . The percentages of pigment production were 61.4% for pyocyanin, 37.1% for pyoverdin and 1.4% for pyorubin. Conclusion: The study demonstrated high rates of antimicrobial resistance markers to most commonly used antibiotics in treatment of P. aeruginosa infections. The majority of the isolates from urine and wound samples carried at least three potential virulence factor genes of algD, lasB and toxA and without any significant relation to their antimicrobial resistance markers.