The purpose of this study was to evaluate the potential of a broad diagnostic approach based on 16S rRNA gene amplification and sequencing for rapid detection of neonatal bacteremia. Subjects and Methods : fifty neonates admitted to the neonatal intensive care units for suspected sepsis, paired blood samples were collected and analyzed for bacterial growth using the BACTEC 9050 instrument and for the bacterial 16S rRNA gene using a PCR assay with subsequent DNA sequencing for bacterial species identification. Results: The positivity rates by blood culture and PCR were 43 (86%) and 44 (88%) positive specimen out of a total of 50 specimen respectively. There was very good agreement between the results of PCR and blood culture methods for diagnosis of neonatal bacteremia (kappa ═ 0.8), taking blood culture as a reference method, the sensitivity, specificity, positive and negative predictive values for PCR were 100% ,75%, 95.5% and 100% respectively after exclusion of candida isolate which detected by blood culture not by PCR as the primer used was 16S r RNA not 18S r RNA needed to identify fungal sepsis. Concerning the time consumed to detect sepsis, blood culture method took more time (up to 5 days) while PCR took less time (