This present study was undertaken for detection of extended spectrum β-lactamases (ESBLS) enzyme genes among clinical isolates of Pseudomonas aeruginosa using phenotypic and molecular techniques. Thirty-four P. aeruginosa isolates from different hospitals in Nsukka and University of Nigeria Teaching Hospital (UNTH), Enugu were screened for the presence of ESBL-encoding genes. Phenotypic screening for ESBL producers was carried out using double disk synergy test and combined disk test. Genomic DNA was extracted from the isolates by modified boiling method. Extracted DNA was amplified by polymerase chain reaction (PCR) using ESBL specific primers namely Bla GES, PER, OXA-50, SHV, CTX-M and TEM. The results revealed that a total of 15 isolates of P. aeruginosa were identified as ESBL producer by phenotypic approaches which exhibited varying degrees of resistance to an array of antibiotics tested. While, the PCR screening revealed that 53.33% (n=8) of the isolates that were phenotypically ESBL positive harboured bla OXA-50 gene. However, the genes that encode PER, GES, SHV, TEM and CTX-M were not found in any of the P. aeruginosa isolates. This study highlights the need to establish antimicrobial resistance surveillance network to determine the appropriate empirical treatment regimen for Pseudomonas infections.