Background: Our aim was to evaluate the influence of methylenetetrahydrofolate reductase (MTHFR) gene polymorphism on maternal risk for Down syndrome (DS) and observe the impact of this polymorphism on folate, homocysteine, and vitamin B 12 concentrations and their association with pregnancy outcome in addition to malformations in DS offspring. Results: The prevalence of MTHFR gene polymorphism at 677 positions in mothers of DS children (DSM) (n = 118) was compared with control mothers (CM) who were age matched with normal children and no history of spontaneous abortion (SA) (n = 118). The MTHFR gene polymorphism was detected using the PCR-RFLP method. MN frequency was measured by CBMN assay and folate; homocysteine and vitamin B 12 were measured using the biochemical analyzer. All statistical analyses were carried out using the chi-square test and t test by using GraphPad Prism 7.0 software. Heterozygous (C/T) genotype was highly significant (p < 0.001) in DSM occurring at 64.4 %, while only 33% CM showed C/T genotype, with an odds ratio of 4.1. Significantly lower levels of folate (p < 0.01), vitamin B 12 (p < 0.001), and higher levels of homocysteine (p < 0.01) were found in DSM compared to CM. The MN frequency was highly significant (p < 0.001) in DSM with C/T genotype when compared to CM. Within DSM, significantly higher (p < 0.001) MN frequencies were observed in DSM with C/T genotype than DSM with C/C genotype. This shows the susceptibility of chromosome malsegregation leading to DS in these women. In addition, the frequency of SA in DSM with C/T genotype was significantly higher (p < 0.01). The DS children showed significantly higher rates of congenital heart defect, preterm birth and low birth weight when mother had C/T genotype. Conclusion: The present study supports the association of MTHFR C677T with DS risk and the above mentioned associated abnormalities in the child. We suggest that identification of MTHFR genotype and adequate folate and vitamin B 12 intake during the preconception and pregnancy period could help protect against congenital malformations and improving pregnancy outcome.