Objectives: We aim to investigate, as a first insight, the presence and rates of high-risk Escherichia coli ST131 clone in Istanbul and evaluate antimicrobial resistance and CTX-M-15 production of ST131 and non-ST131 isolates. The use of MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry) to detect E. coli ST131 clone is also evaluated. Materials and Methods: A total of 203 extended-spectrum beta-lactamase (ESBL)-producing urinary isolates from a training hospital in Istanbul were investigated. Detection of E. coli ST131 was done by MALDI-TOF MS and real-time PCR melting curve analysis. The presence of CTX-M and CTX-M-15 beta-lactamases was investigated by PCR and sequence analysis. Results: Of the 203 isolates, 81 (39.9%) and 75 (36.9%) isolates were identified as ST131 clone by PCR and MALDI-TOF MS, respectively. Resistance to ciprofloxacin was significantly higher among ST131 isolates. A total of 169 (83.5%) isolates produced CTX-M beta-lactamase, of which 72 (43%) were CTX-M-15. The production of CTX-M and CTX-M-15 were significantly higher among ST131 isolates. Conclusions: We have demonstrated, for the first time, high rates of ST131 clone among ESBL-producing E. coli isolates in Istanbul, a region with high rates of resistance to third-generation cephalosporins and fluoroquinolones. Further investigation of this high-risk clone and its contribution to high antimicrobial resistance in Turkey is essential. MALDI-TOF MS is a useful tool for detection of high-risk clones and associated resistance patterns, simultaneous to bacterial identification.