Bloodstream infections (BSIs) and especially central line associated bloodstream infections (CLABSIs), are increasing globally as an important cause of morbidity and mortality. The condition can be life threatening in critically ill patients in the intensive care units (ICUs) of the hospitals. There are serious concerns about the rapid increase of multidrug resistant Gram negative bacteria (MDR-GNB) such as Escherichia coli, Klebsiella pneumonia and Acinetobacter baumannii as the major causes of bloodstream infections. The aim of the study was to emphasize on the significance of MDR-GNB, determine their antibiotic susceptibility pattern, genotypic identification as well as the detection of some virulence genes. Blood specimens were collected from 231 bacteremic patients admitted to different ICUs at three tertiary care hospitals in Egypt. Recovered bacterial isolates were subjected to biochemical identification using conventional methods. Antimicrobial susceptibility testing was performed on Gram negative isolates using both Kirby-Bauer disc diffusion method and Vitek-2 system. Thirty MDR-GNB isolates were selected and screened for the presence of 16S-23S rRNA, UIDA and OMPA genes using PCR assays to confirm their identification. Also, the thirty isolates were screened for the presence of East-1, Cnf-1, IutA and FyuA genes.  A total of 185 clinical isolates were recovered from 185 positive blood specimens. Laboratory examination of the positive blood culture specimens revealed that 120 (64.4%), 56 (30.2 %) and 9 (4.8%) were Gram negative, Gram positive and Candida spp., respectively. Thirty MDR-GNB were selected and their antimicrobial susceptibility was compared using the conventional and the Vitek-2 system. For the K. pneumoniae isolates 100% Categorical agreement (CA)was detected for Ceftriaxone, Cefepime, Gentamicin, Amikacin and Tetracycline. For the E. coli CA of (100%) was seen for Ampicillin+Sulbactam, Cefepime, Amikacin, Ciprofloxacin and Tigecycline. categorical agreement (100%) was seen with the tested antibiotics for A. baumannii. Genotypic confirmation of the selected MDR isolates indicated the presence of genes used for identification in all of them. As for the harboring of virulence genes, EAST-1 gene has only been found in 13.3% of the isolates. CNF-1 gene has been identified in 66.6% of the isolates,IutA gene has been found in 96.6% of the isolates and FyuA has been found in 90% of the isolates. Gram negative pathogens were found to be the major bacteria causing bloodstream infection. Vitek-2 system provide accurate antimicrobial susceptibility testing results. CNF-1 toxin production related gene was found to be prevalent in most of the isolates. IutA and FyuA siderophore formation related genes were found to be prevalent in most of the isolates.