Abstract: The present study was performed to isolate, characterize and evaluate the prevalence of Pseudomonas aeruginosa (P. aeruginosa) in 600 samples (environmental and clinical) at Upper Egypt. Antibiotic sensitivity, biotyping and molecular identification of P. aeruginosa isolates were determined, as well as hemagram was done on all positive clinical cases. Out of examined 200 environmental samples, 42 P. aeruginosa strains (21%) were isolated. Out of examined 400 clinical specimens, 70 and 16 P. aeruginosa strains (35 and 8%) were isolated from sputum and urine, respectively. The Multi-Drug Resistant (MDR) P. aeruginosa isolates gave resistance to a number of antimicrobial types ranged from 4/14-10/14. The total antimicrobial resistance profile of P. aeruginosa isolated from environmental and clinical samples ranged from 3.1% (colistin) to 95.3% (cephalothin, cefuroxime and cefoxitin). The total mean of MIC of tested antimicrobials versus P. aeruginosa (n = 128) ranged from 1.13±0.02 μg mL-1 (colistin) to 24.7±2.1 μg mL-1 (ceftriaxone). The prevalence of P. aeruginosa between environmental and clinical samples showed significant correlation. Moreover, significant correlation found in total antimicrobial resistance profile between the tested antimicrobials except imipenem and aztreonam shown non significant (p>0.05). PCR analysis of P. aeruginosa indicates that species specific signature sequences were present in 100% of the tested isolates. The hospital environment and healthcare personnel could serve as potential reservoirs of P. aeruginosa in the study locality. The obtained results may help in prevention and control strategies of P. aeruginosa infection in both the hospital and the community.