Background: Sepsis is one of the leading causes of morbidity and mortality in children worldwide, blood culture is an imperfect gold standard since it's insensitive and slow. A modern microbiological diagnostic work-up increases the sensitivity and enables to directly identify fastidious, slow-growing, unculturable, or nonviable bacteria. Objective: This study aimed to evaluate the performance of 23S rDNA PCR assays as a rapid detection method for diagnosis of sepsis in children with suspected bacteremia in comparison with automated blood culture. Methods: The study included 93 children (51 male and 42 female). They were under empirical antibiotic therapy, their ages ranged from 2 days to 6 years old, fulfilling sepsis - 3 criteria using QSOFA to identify suspect cases and SOFA to assess organ affection. Blood culture was positive in 63 samples and negative in 30 samples. PCR for 23S rRNA gene was done for 50 cases of them (20 positive blood culture and 30 negative blood culture) used as a study group. Results: The present study showed that, out of 20 blood culture positive samples, 20 (100%) were found positive by 23S rRNA gene PCR assays. Positive PCR results were obtained in 29 samples of the 30 blood culture negative samples. Accordingly, the resulting detection rate of 23SrRNA gene PCR (98%) was higher than blood culture (40%) in all fifty samples. Conclusion : The real time PCR of 23S rRNA gene provide rapid and reliable detection of bacterial pathogens directly from patient blood samples which helps to optimize antimicrobial therapy.