Detection of AmpC and ESBL producing P. aeruginosa by phenotypic methods is challenging, especially in low-income countries such as Pakistan. Therefore, a molecular method was developed for rapid detection of these resistance markers. A total of 303 clinical samples were collected from intensive care units (ICUs) of the Jinnah postgraduate medical centre (JPMC) Karachi, Pakistan. The isolates were identified by traditional and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF-MS). Isolates were phenotypically analyzed for AmpCs and ESBL by D-test and by double disc synergy, respectively. The Check MDR CT103 XL and PCR techniques were used for the detection AmpCs and ESBLs. Out of 303 isolates, 148 (48.8%) were P. aeruginosa. The resistance pattern of P. aeruginosa against piperacillin, cefatizidime and cefepime was 59.4%, 64.8% and 59.4% respectively. More than 60% isolates were resistant to aminoglycosides and ciprofloxacin. All (148) strains were found sensitive to colistin. Phenotypic ESBL prevalence was 8.8% whereas genotypic resistance was 29.1%. bla was the most prevalent ESBL. Although 25.67% of P. aeruginosa isolates were positive phenotypically for AmpC, microarray (Check-MDR) analysis did not detect chromosomally located AmpC in any of the isolates. VE