Pseudomonas (Ps.) aeruginosa is one of the most common pathogens causing nosocomial infections. This pathogen causes several infections including urinary tract infection (UTI), wound infection, pneumonia, bacteremia,…etc. Immunocompromised patients and patients in intensive care unit are at high risk of acquisition of infection, in addition multidrug resistant Ps. aeruginosa isolates had been characterized. This study was planned to determine the frequency of Ps. aeruginosa in nosocomially infected patients in Assiut university hospital and to type isolated strains. In this study, 677 samples collected from 366 nosocomially infected patients admitted to different hospital wards at Assiut University Hospitals, including chest, trauma, neurology, internal medicine, post operative and pediatric ICUs, trauma and hematology units. Identification of bacterial strains was done by cultural and biochemical tests. Biotyping analysis for isolated strains was done using API 20NE. In our study, a total of 30 (8.2%) Ps. aeruginosa strains were isolated. Four API codes profile for Ps. aeruginosa isolates were identified, the isolate with API code profile 1154575 was more frequent distributed in Assiut University Hospital.