Background: Pseudomonas aeruginosa is one of the leading causes of hospital acquired in lections. lt is one of the most common antimicrobial resistant pathogens commonly associated with bacteremia and sepsis in intensive careunits (TCU), cystic fibrosis (CF), pneumonia, urinary tract, bum and wound infections. Data on the prevalence and a ntimicrobi al resistance profile of this pathogen from different clinical samples is limited in the present study area Objective: The objective of this study was to detennine the prevalence of Pseudomonas aeruginosa, its antimicrobial susceptibility profile and associated factors from different clinical specimens at Debre Tabor Comprehensive Referral Hospital (DTCRH). Methods: A cross-sectional snidy was conducted from IO May 2022 to 15 July 2022 at DTCRH. Participants were recniited using convenience sampling method. The patients' socio-demographic and clinical data were collected by using structured questionnaire. Clinical specimens (blood, wound swab, urine and sputum) were collected based o n the standard microbiological procedures. Conventional bacteriological culture was done and the isolate were identified by usi.ng panels of biochemical tests. Antimicrobial susceptibility testing was done using disk -diff usion method as per tbe Clinical and Laboratory Standard Institute (CLSJ) guidelines. Data were entered and analyzed using SPSS version 25 statistical software. Results: Three hundred eighty four participants were included, of which the majority 238(62%) were male. The mean ±SD of participants' age was at 35.36 ±16.7 years. The prevalence of P.aeruginosa was 74 (19.3%). The detection of the isolate was different based on the sample types. It was 0% from sputum while the prevalence was 54.5% from wound swab. P.aeniginosa isolates were found res istan t for gentamicin 62.2%, ceftazidime 51.4%, cefepime 50%, amikacin 29.7%, imipenem 28.4% and ciprofloxacin 14.9%. The rate of multi-drug resistance (MDR) of these isolates was 45.9% and suspicious extreme-d rug resistance (XDR) was 9.5%. Being inpatient and wound swab sample were significantly associated with the detection of P.aeruginosa (p<0.05). Conclusion: The overall prevalence of P.aeruginosa was 19.3%. High prevalence of P.aeruginosa was identified from wound swab (54.5%). In this study high rate of MDR was identified, which was 45.9% and suspicious XDR was 9.5%. Being inpatient and pus sample were significantly associated with the detection of P.aeruginosa. Even though the prevale nce of P.aeruginosa was medium in this study compared to the previous studies, the rate of MDR was very alarming. Therefore. actions to minimize antimicrobial resistance should be strengthened by different stakeholders. Keywords: Pseudomonas aeruginosa, clinical specimens, prevalence, Antimicrobial susceptibility profile.