Abstract: Background and Objective: Pseudomonas aeruginosa (P. aeruginosa) is considered one of the most dangerous opportunistic bacteria worldwide, live in hospitals and other environments such as sewage water and soil and cause different infections include pneumonia. The main aim of this study was to investigate antibiotics susceptibility pattern and genotypic detection of virulence genes in P. aeruginosa isolates from clinical and environment sources. Materials and Methods: A total of 120 P. aeruginosa isolates were collected from two sources, 60 isolates from sputum of patients with pneumonia and 60 isolates from sewage water. Antibiotics susceptibility testing was done according to Kirby-Bauer method. Polymerase chain reaction technique was performed to detect the prevalence of six virulence-associated genes. Results: All 120 isolates (100%) were resistance to amoxicillin. While, imipenem was the best efficient antibiotic against clinical and sewage water isolates. Fifty five clinical isolates (91.6%) and 41 sewage water isolates (68.3%) were multi-drug resistance (MDR). The Pap A, Fim H, feoB, iutA, hly and kpsMTII genes prevalence in clinical isolates with percentages 91.6, 100, 80, 95, 20 and 100%, respectively, while the same genes prevalence in sewage water isolates with percentages, 51.6, 68.3, 41.6, 35, 3.3, 5 and 41.6%, respectively. Conclusion: This study suggested that there was positive correlation between the high prevalence of virulence-associated genes and increase of antibiotics resistance in P. aeruginosa isolates.