Objective: This study determined the prevalence of ESBL genes amongst Klebsiella species isolated from patients’ specimens attending a Tertiary Teaching Hospital, in Ile-Ife, Southwest Nigeria. Methods: A cross sectional study of presumptive isolates of Klebsiella (n=180) were collected, after ethical approval in the microbiology laboratory. Isolates were identified to species level by conventional biochemical tests and MicrobactTM 24E Identification Kits. Antibiotic susceptibility testing was performed by the Kirby Bauer’s disk diffusion method. ESBL production was detected by the double disc synergy and ESBL genes by the multiplex PCR protocol. Results: The Klebsiella species identified were Klebsiella pneumoniae 95.6%, Klebsiella oxytoca 3.3%, Klebsiella ornithinolytica 0.6% and Klebsiella terrigena 0.6%. The prevalence of ESBL genes among the Klebsiella isolates was 47.2% , and the most common ESBL gene was blaSHV (38.9%), also 75% of the study isolates had MAR index greater than 0.2. Conclusions: The study establishes the prevalence of Extended Spectrum Beta-Lactamase producing Klebsiella sp in the hospital and identified the most prevalent ESBL genes circulating as blaSHV followed by blaTEM and blaCTX-M in this environment. This underscores the need for regular and continuous surveillance of antimicrobial resistance trend as a strong component of the antibiotics stewardship and infection prevention and control programs in the hospital. Keywords: Antibiotic Resistance; Klebsiella; ESBL.