Background: High-mobility group box 1 (HMGB1) has important role in a variety of diseases, including neonatal sepsis (NS). The aim of this study is to evaluate the clinical importance of HMGB1 genetic expression in NS demonstrating its diagnostic value and detecting the effect of its genetic expression on the disease outcome. Patients and Method: This study was carried out on 100 neonates; 28 neonates with clinical sepsis and had positive blood cultures (confirmed), 22 neonates with clinical sepsis but had negative blood cultures (suspected) and 50 healthy non-infected newborns age and sex matched as controls.Results: Expression of HMGB1 in newborns with confirmed NS and those with suspected NS were higher than healthy controls. Also, it was higher in newborns with confirmed NS than those with suspected NS. The sensitivity and specificity of the diagnostic value of HGMB1 were higher than that of C-reactive protein (CRP) and hematological score (H score). Assessment of the HMGB1 for prognosis of the disease was evaluated by univariate analysis (p=<0.002) and logistic multivariate regression analysis (p=<0.023). The univariate analysis showed that CRP, H score and HMGB1 are significantly predicting the prognosis of NS, while the multivariate analysis showed that only HMGB1 significantly predict the prognosis of NS. There were significant positive correlations between HMGB1 gene expression and APGAR score at 5 minutes, hemoglobin level, H. score and CRP titer in confirmed N Conclusion: HMGB1 is a possible diagnostic and prognostic marker of neonatal sepsis (NS).