Background: Extended-spectrum β-lactamase (ESBL) producing uropathogens has become prevalent worldwide. E. coli O25b-ST131 clone, associated with blaCTX-M-15, has been reported from many parts of the world and is frequently associated with multidrug resistance. Thus far, there are no reports about this clone in Bangladesh. The objective of this study was to investigate ESBL producing uropathogens and to survey the prevalence of E. coli O25b-ST131 clone among ESBL positive E. coli isolates. Methods: From symptomatic urinary tract infection cases, a total of 800 urine samples were collected. Bacterial identification and antimicrobial susceptibility testing was performed using established methods. Screening of ESBL producers was done using the disk diffusion method. Screening positive isolates were phenotypically confirmed by double disk synergy (DDS) test. Genes encoding ESBLs (blaCTX-M-15, blaOXA-1) were identified both by PCR and DNA sequencing. Phenotypic positive ESBL producers were also studied by PCR for existence of class 1 integron. Subsequently, O25b-ST131 clone was identified by allele specific PCR. Results: Of 138 gram-negative uropathogens, 45 (32.6%) were positive for ESBLs. ESBL producers showed high frequency of antimicrobial resistance except imipenem. Among 45 ESBL producers, 36 (80%) produced blaCTX-M-15, 18 (40%) produced blaOXA-1. Fifteen (33.3%) strains simultaneously produced both blaOXA-1 and blaCTX-M-15. Class 1 integron was present in 30 (66.7%) isolates. Of the 31 blaCTX-M-15 positive E. coli, 22 (71%) were positive for E. coli O25b-ST131 clone and all (100%) belonged to B2 phylogenetic group. Conclusion: Rising antimicrobial resistance among uropathogens, and especially the emergence of blaCTX-M-15 positive E. coli O25b-ST131 clone in Bangladesh has provided urgency to the development of novel preventive and therapeutic strategies.