Background: Prematurity is a significant global health concern, often associated with neurodevelopmental challenges. Epigenetic regulation of stress-related genes, such as SLC6A4, influences neonatal stress adaptation and developmental outcomes. Aim: This study quantified and compared DNA methylation levels at 13 CpG sites in the SLC6A4 promoter region between preterm and term neonates at birth (D0), the fifth day (D5), and the thirtieth day (D30) of life.​​​​​​​ Methodology: A cohort of 46 preterm and 49 full-term neonates was analyzed. Blood samples were collected at D0, D5, and D30, and DNA methylation levels were assessed using bisulfite conversion and pyrosequencing.​​​​​​​ Results: Significant differences in SLC6A4 methylation were observed. At D0, CpG 12 and CpG 13 showed higher methylation in preterm neonates, with CpG 13 methylation remaining significantly higher on D5. Intriguingly, CpG 9 showed lower methylation in preterm neonates on both D0 and D5. Distinct patterns emerged across very preterm and extremely preterm subgroups over time. Conclusion: This study reveals significant differences and dynamic changes in SLC6A4 methylation in preterm neonates, highlighting the impact of prematurity and early-life stress on the epigenome. These findings enhance our understanding of the epigenetic mechanisms shaping neurodevelopment and stress adaptation in neonates.