Background: The risk factors of neonatal mortality are complex, this health indicator is relatively improved slowly and its decreasing trend is not as great as other health indicators such as infant and under 5 years children mortality rates. This study aimed at identifying the risk factors of neonatal mortality in Gitwe District Hospital, based on observational casecontrol study design. Methods: A total of 3782 neonates, who took post-neonatal care at Gitwe Hospital (Rwanda), were enrolled from January 2013 to July 2014; and we found 62 died neonates. By using systematic random sampling, 186 controls were selected among survivors. Maternal and neonatal data were collected and the outcome of interest was neonatal mortality. Multiple logistic regression models were used to examine the association between studied factors and neonatal mortality. Results: The factors that highly associated with neonatal mortality(5 < aOR < 25, P ≤ 0.001) were grandmultigravida, HIV+, incomplete antenatal care visits, eclampsia, delivering at home, prolonged labor duration, and lower 1st minute Apgar score. Other factors were less associated with neonatal mortality (1 < a OR < 5, P ≤ 0.05), those are lack of health insurance, early childbearing (<20 years), preterm birth, and asphyxia. Lower birth weight and lower birth spacing (aOR < 1, P ≤ 0.01) showed the negative association with neonatal mortality. Conclusion: This study has identified some maternal and neonatal factors which should be used to educate health care services providers, administrators and as the whole the community so as to reduce neonatal death rates.