The potential utility of real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal sepsis. Turk J Pediatr 2019; 61: 493-499. The purpose of this study was to evaluate the efficacy of real-time polymerase chain reaction (PCR) of the 16S rRNA gene in diagnosis of neonatal sepsis and compare it with conventional blood culture. A total of 150 infants were enrolled in this prospective study. The infants were classified into two groups: sepsis group (n=100) and control group (n=50). Blood samples for complete blood count, C-reactive protein, procalcitonin, serum-amyloid A, blood culture and PCR were obtained before initiating antibiotic treatment. Eight specific probes were used to perform PCR analysis for detection of 8 different microorganisms. The positivity rates of blood culture and PCR were found as 11% and 3%, respectively. The diagnosis of neonatal sepsis by PCR revealed a 16.6 % sensitivity, 97.8 % specificity, 33.3% positive predictive value and 94.8% negative predictive value compared with the blood culture. This study showed a low sensitivity of PCR of the 16S rRNA gene in the diagnosis of neonatal sepsis. This may be associated with the identification of rare microorganisms in the blood culture that were not included to PCR analysis. Implementation of all suspectible microorganisms into PCR assay may increase the sensitivity of 16S rRNA gene PCR in diagnosis of neonatal sepsis.