Background: Pneumothorax is more common in neonatal period than the rest of the life. In term infants, Pneumothorax is usually asymptomatic. However, premature neonates usually have symptoms particularly when they need mechanical ventilation. The aim of the present study was to investigate the prevalence and risk factors of Pneumothorax among premature infants.Methods: This cross-sectional study was carried out in Alzahra and Shahid Beheshti University hospitals, Isfahan, Iran, in 2010. Neonates with gestational ages of 26-37 weeks hospitalized in neonatal intensive care unit (NICU) were included. Demographic features including gestational age, sex, and birth weight were collected. All neonates who had sudden changes in breathing status were evaluated for Pneumothorax using chest X-ray (CXR). Data were analyzed using Student-t and X2 tests.Findings: Among 303 studied neonates, 29 (9.6%) were suffered from Pneumothorax. Birth weight, gestational age, number of pregnancy and mothers’ chronic diseases were significantly different in two groups (P<0.001 for all). Chest compression, ventilation with bag and mask and needing intubation were statistically different between the two groups, too (P<0.001).Conclusion: Premature neonates are at higher risk for Pneumothorax due to lower birth weight, lower gestational age, higher number of mother’s pregnancy, mother’s diseases during pregnancy; also, interventions such as chest compression, ventilation with bag and mask and intubation procedure is more needed for them. Thus, many cautions during medical intervention must be done for prevention of Pneumothorax.