Background: Sepsis is a major cause of preventable neonatal mortality and morbidity around the world. We evaluated increase in culture positivity rate of two blood cultures taken simultaneously from different sites as compared to single blood culture in diagnosing sepsis in neonates. Material and methods: This was a descriptive cross sectional study done in NICU of a tertiary-care centre from September 2017 to June 2018 on 200 neonates. Two blood cultures were drawn from two different peripheral veins in neonates with suspected sepsis. Primary outcome measure was increase in culture positivity rate with use of two blood cultures. Results: Thirty eight (19%) neonates had positive diagnostic blood cultures from both sites. Twenty five (12.5%) neonates grew organism from one site but not from the other site. 137 (68.5%) patients had sterile cultures from both sites. Overall, first culture positivity was seen in 50 cases (25% first culture positivity rate). On adding the result of second culture, the yield of positivity increased to 63 cases (31.5% total culture positivity rate). Thus adding on second culture, yield increased in 13 cases but it was statistically not significant (6.5%) (CI: -2.3%-15.3%) (p=0.148). Conclusion: There is no role of sending simultaneous two site blood cultures in the diagnosis of neonatal sepsis.