Background: The spread of carbapenem resistance among Enterobacteriaceae have become a problem for healthcare facilities worldwide. Community and hospital-acquired infections caused by these bacteria have been associated with significant morbidity and mortality with limited treatment options. Rapid detection of carbapenem resistant Enterobacteriaceae (CRE) is important for infection control. Objectives: To detect the prevalence of carbapenem resistant Enterobacteriaceae (CRE) species and determine their antimicrobial susceptibility profile using the Vitek 2 system and the presence of carbapenemases genes using Multiplex PCR. Methodology: Various clinical samples were collected from 469 patients from Sohag University Hospitals in the period between August 2016 and April 2018, CRE isolates were identified by conventional methods and antimicrobial susceptibility testing using disc diffusion method and also performed by Vitek 2 automated system, Multiplex PCR was used for detection of carbapenemases genes as blaKPC, blaVIM, blaIMP, blaNDM-1 and blaOXA-48. Results: The prevalence of carbapenem resistant Enterobacteriaceae (CRE) species was 19.9%, Klebsiella pneumoniae was the most common species (51.4%), Escherichia coli (28.6%), Enterobacter aerogenes(8.6%) and Acinetobacter baumannii (5.7%). Vitek 2 system identified CRE isolates with 82.7% sensitivity, 98.6 % specificity and 90.6% diagnostic accuracy 25.7% of CRE strains were isolated from the internal ICU and 20 % from Chest Department, and mostly isolated from urine(40%) and from endotracheal tubes swabs(28.6 %) 77.1 % of CRE isolates contained carbapenemases genes, 62.1 % were blaKPC positive, 20.7 % were blaVIM-positive, 3.4 % were blaNDM-positive, 13.8 % were blaOXA-48-positive and none was blaIMP-positive. Conclusion: Conventional methods supported by Vitek 2 system is a valuable method for identification of CRE species, the detected carbapenemases genes in this study indicate that carbapenem resistance is spreading in Egypt and support the use of molecular methods for the rapid detection of CRE for successful implementation of infection control measures. We recommend routine testing to determine carbapenem resistance in Enterobacteriaceae in health facilities in Egypt.