Background and Aim: The risk factors of the Neural Tube Defects (NTD) have been previously described but there are ethnic and geographical variations. Data from the Iranian population is still scarce. The objective of the current study was to investigate the NTDs risk factors in a large sample of Iranian patients admitted to a single center. Methods and Materials/Patients: This case-control study was performed within five years from 2012 to 2017 in Namazi Hospital of Shiraz, a tertiary referral center for neonatal anomalies in the south of Iran. One hundred newborns with NTDs were included in the study as the case group and 200 healthy newborns as the control group. We recorded the baseline characteristics including the maternal variables (age, weight, height, previous pregnancy and gravidity, gestational age), newborn information (birth weight, clinical diagnosis, clinical findings in the examination, and clinical findings in radiologic test) and medical history of the perinatal period. Results: The baseline characteristics of the mothers were matched in both groups. NTDs were associated with lower folic acid intake during pregnancy (66% vs. 78%; P=0.030; OR 95% CI=1.82) and before pregnancy (P=0.002; OR95% CI=2.36). The prevalence of NTD was significantly higher in patients who lived in hot climates (P=0.001). Conclusion: Taking adequate folic acid supplements before and during pregnancy can reduce the risk of NTDs in the Iranian population. Hot climate zones were associated with an increased risk of NTDs in Iran.