Background: Few studies from developing countries have examined sensitivity, specificity, positive and negative predictive values of routine surface cultures. Objectives: The purpose of the study was to determine sensitivity, specificity, and positive predictive value (PPV) of skin cultures among preterm neonates admitted to Dhaka Shishu Hospital, Bangladesh. Methods: The study was nested within a prospective, randomized, controlled trial of emollient treatment in Dhaka Shishu Hospital, Bangladesh. A total of 497 preterm infants <33 weeks gestational age and <72 h of chronological age were enrolled, and the sensitivity, specificity, and PPV of skin cultures were analyzed among 3,765 blood-skin culture pairs, wherein the skin culture was obtained within 13 days before the blood culture. Results: Overall sensitivity, specificity, and PPV were 16, 38, and 5%, respectively. PPV during Klebsiella pneumoniae outbreaks was about 9%, and the inguinal site had the highest PPV (6%) among the three skin sites. Acinetobacter spp.- and K. pneumoniae-specific PPVs were 28 and 23%, respectively. PPV was <2% for Candida spp., Enterobacter spp., and Salmonella spp. Conclusion: Routine skin culture is inefficient in predicting the pathogen responsible for sepsis among premature neonates, even in a developing country setting, where the burden of bacterial infection is relatively high. Skin cultures are also of limited utility during K. pneumoniae outbreaks, and are not recommended.