Extended spectrum beta-lactamases (ESBLs), mainly produced by Gram-negative bacteria, hydrolyze quinolones leading to multi-drug resistance challenges encountered in clinical settings. Detection of ESBLs will help in treatment option. This study, therefore, investigated the presence of ESBL encoding gene by polymerase chain reaction (PCR) in Enterobacteriaceae isolated from clinical samples. A total of 100 clinical samples submitted for routine microbiological analysis were obtained and examined for Enterobacteriaceae using standard microbiological methods. Bacterial identity confirmation was performed by API 20E kit and PCR using ropB specific primers. Sensitivity test was determined using agar diffusion method while ESBLs were confirmed by PCR. Thirty (30) Enterobacteriaceae isolates consisting of Escherichia coli, Klebsiella pneumoniae, Salmonella enterica and Serratia were obtained from 100 clinical samples. The rpoB gene was confirmed in all the 30 isolates. All isolates were resistant to the all tested antibiotics (ceftazidime, cefuroxime, gentamicin, cefixime, ofloxacin, amoxicillin/clavulanate, nitrofurantion, and ciprofloxacin). The PCR-based ESBLs confirmation revealed that 21 (70%) of the 30 isolates possessed gene for SHV type enzyme while TEM and CTM-X types were not amplified. Conclusions The study indicates a high level of multi-drug resistant ESBL-producing Enterobacteriaceae in clinical setting in Lagos. Hence, a proactive antibiotic surveillance system is urgently needed to curtail spread of resistant bacteria in the community.