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Background: Extended spectrum beta-lactamases are produced by Gram-negative bacteria and most strains producing them belong to the family Enterobacteriaceae. The greatest challenge with ESBL-producing Enterobacteriaceae is their propensity to acquire multidrug resistance traits. This study aimed at determining the prevalence of ESBL-producing Enterobacteriaceae among selected tertiary hospitals in south-eastern Nigeria. Methods: A total of 400 Enterobacteriaceae isolates were obtained from patients attending five selected tertiary hospitals and were identified to species level by Gram staining and conventional biochemical tests. Screening for ESBL production was determined by the Kirby-Bauer disk diffusion method using 30µg disk of ceftriaxone, cefuroxime, cefpodoxime, ceftazidime, and aztreonam while confirmatory test was done using combination disk test based on the 2016 CLSI guidelines. Results: The prevalence of ESBL production among Enterobacteriaceae isolates from selected hospitals in southeast Nigeria is 61.5% (246 of 400). Among the isolates obtained, the highest prevalence was observed in Klebsiella oxytoca (100%) while the least prevalence was seen in Morganella morganii (50.0%). Escherichia coli and Klebsiella pneumoniae had rates of 61.8% and 62.3% respectively. Among the States of the south-east Nigeria, selected hospital in Ebonyi had a prevalence of 83.5%, Abia 63.6%, Anambra 61.5%, Enugu 51.7% and Imo 36.5%. The prevalence of ESBL-producing Enterobacteriaceae differ significantly between the States (p=0.000). Conclusion: ESBL-producing Enterobacteriaceae strains have been isolated from different participants, from the selected tertiary hospitals in south-eastern Nigeria. Therefore, we report a high prevalence of ESBL-producing Enterobacteriaceae in south-eastern Nigeria. [ABSTRACT FROM AUTHOR]