Background: Rapid identification of infection caused by Klebsiella pneumoniae carbapenemase producing bacteria, is critical for the beginning of appropriate antimicrobial treatment and ending their spread. Objectives: to study and evaluate RT-PCR technology as a rapid method to directly detect KPC-producing Enterobacteriaceae in positive blood culture bottles. Methodology was: cross sectional study, conducted at Benha University Hospital. 253 blood culture bottles were incubated in the BD BACTEC™ 9050 Blood Culture System for up to 5 days, and Gram staining was done when the bottles were identified as being positive by the Bactec system. All Gram-negative pathogens were subcultured and identified Antimicrobial susceptibilities to meropenem and imepinem were tested by disc diffusion and interpreted according to The Clinical and Laboratory Standards Institute guidelines 2014. Klebsiella pneumoniae carbapenemase producing enterobacteriaceae in positive blood culture bottles was identified by Real time PCR. Results: out of 253 specimens,235 were positive, 186 (79.1%) were Enterobacteriaceae, 57 isolates (30.6%) of all Enterobacteriaceae isolates were resistant by disc diffusion test, and real-time Polymerase chain reaction detected 55 isolates (29.6%) as positive for the presence of Klebsiella pneumoniae carbapenemase gene. The sensitivity of PCR was 96.5%, specificity was 100%, PPV=100%. NPV= 98.5%, and diagnostic accuracy was 98.9%, in comparison with disc diffusion as a gold standard test. Conclusion: real time Polymerase chain reaction is a useful, rapid, sensitive, and specific tool to detect Klebsiella pneumoniae carbapenemase directly in positive blood culture bottle.