Background: Globally, there is a changing trend in the antibiotic susceptibility pattern of Gram-negative uropathogens to the conventional drugs used in the treatment of urinary tract infections due to the production of extended-spectrum beta-lactamases (ESBLs). Aim: This study aimed to determine ESBL production and antimicrobial susceptibility pattern in uropathogens. Materials and Methods: Five hundred urine samples submitted to the Medical Microbiology Department of Ahmadu Bello University Teaching Hospital from January to June 2012 were analyzed by conventional methods. Modified standardized Kirby-Bauer disc diffusion method was used for antimicrobial susceptibility testing. ESBL production by Escherichia coli and Klebsiella pneumoniae isolates was screened for using the Clinical and Laboratory Standards Institute guidelines 2012 and confirmed by the double-disc synergy tests. Results: Five hundred samples were analyzed. Of these, a total of 175 Gram-negative isolates were obtained. Isolation rates were E. coli – 56%, K. pneumoniae – 20%, Proteus mirabilis – 16%, and Pseudomonas aeruginosa – 4%. ESBL production was observed in 34.3% of all the isolates. Fifty percent (50%) of E. coli and 40% of K. pneumoniae were identified as ESBL producers and were found to be resistant to multiple antimicrobial agents. Imipenem and nitrofurantoin had sensitivity of 100% and 70%, respectively, while susceptibility to ciprofloxacin and gentamicin was low at 35% and 30%, respectively, although 96% sensitivity was observed with amikacin. ESBL producers and nonproducers showed a high level of resistance of over 95% to ampicillin, amoxycillin, and trimethoprim-sulfamethoxazole. Conclusion: This study found a high rate of ESBL production (34.4%) among uropathogens with multidrug resistance. Clinical microbiology laboratories should routinely incorporate ESBL detection methods in their laboratory procedures for continuous surveillance of multidrug-resistant isolates and antibiograms to guide empirical therapy.