Prenatal alcohol exposure (PAE) is considered as a risk factor for the development of fetal alcohol spectrum disorders (FASD). Mounting evidence suggests that PAE affects epigenetic mechanisms and alters the normal differentiation and development of neural stem cells (NSC) in fetal brain. DNA methylation, as an important epigenetic mechanism, contributes to several biological processes during brain development and undergoes significant changes following PAE. In addition, recent studies suggest that DNA methylation is a considerable biomarker for the early diagnosis of FASD. However, effects of PAE are dependent to a variety of factors such as sex and strain of studied subjects. In this regard, we aimed to investigate whether sex and strain play a determinant role in the effects of chronic ethanol exposure on DNA methylation machinery. In addition, based on our previously studied RNA-Sequencing data, we tested the validity of three potential biomarkers (Dcc, Scn3a, and Sptbn2), which were suggested by IPA (Ingenuity Pathway Analysis) software. To do this, we applied a standard and controlled in vitro model system, in which we treated male and female differentiating NSC (obtained from the forebrain of CD1 and BL6 embryos at the embryonic day 14.5) with chronic ethanol exposure (70 mM) for 8 days. Our results showed that chronic ethanol treatment induced global DNA hypomethylation and altered the components of DNA methylation machinery in a sex and strain-specific manner. These alterations in DNA methylation status were associated with altered expression of glial markers CNPASE, GFAP, and OLIG2 in CD1 (but not BL6) cells. We also found that Dcc, Scn3a, and Sptbn2 and their corresponding proteins were altered by ethanol exposure, but may not be true candidate biomarkers for FASD biomarkers because their alterations were not consistent in all experimental groups. In conclusion, the effects of ethanol on DNA methylation of differentiated NSC depended on sex and strain of cells, and DNA methylation alone may not be a suitable biomarker for FASD without consideration of sex and strain. In addition, the effects of ethanol on NCS fate commitment were only observed in CD1 cells and not BL6 cells. Taken together, the results of this work provided evidence that chronic ethanol exposure affects DNA methylation and cell fate commitment in differentiated NSC, but in a sex and strain-specific manner. My results also revealed that either DNA methylation and/or candidate biomarkers (Dcc, Scn3a, and Sptbn2) alone may not be suitable biomarkers for FASD.