The rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE) all over the world represents a matter of concern. This study aims to investigate the prevalence of CRE in clinical isolates recovered from Al Kasr Al-Ainy hospital in the period between August 2015 and February 2017. The isolates were identified by conventional methods and Maldi-TOF spectroscopy. Phenotypic identification of CRE was carried out by Modified Hodge test and genotypic characterization for Extended-Spectrum Beta-Lactamase (ESBL) and CRE genes was also performed. Carbapenemase activity of CRE isolates was confirmed in 46% of the isolates of which, blaNDM-1 and blaOXA-48 were detected in 75%, 59% of the isolates respectively, while blaVIM was detected in 2.3% only. However, blaIMP and blaKPC were not detected in any isolate. All CRE isolates carried at least one ESBL gene, 95.4% of CRE isolates had blaCTX-M-15, 88.6% blaTEM-1, and 68.2% blaSHV. The blaSHV gene showed different alleles in the CRE isolates. All the isolates were sensitive to polymyxin B and colistin while only 36.4% were sensitive to tigecycline. Consequently, microbiologists and clinicians should implement the necessary control measures to prevent the spreading of these resistant bacteria.