Many international studies indicated that the polymorphisms of some genes disturbed the folate homocysteine (Hcy) metabolism and increased the vulnerability to Down syndrome (DS). We aimed to measure the serum levels of folate and Hcy in DS children and compare the levels with age and sex-matched apparently normal healthy children. We also aimed to study the A80G polymorphism of the gene reduced folate carrier (RFC1) in the DS children as a risk factor. Forty children with DS (24 were boys, and 16 were girls) with the age range between 5-13 years, and 26 normal healthy children (16 boys and ten girls) were included in this study. The results show that the highest genotype in the control group was AG (53.85%) followed by AA and GG (30.77% and 15.38%) respectively. The genotype percentages in children with DS were (50, 35,and 15) for AG,AA, and GG respectively. There was no statistically significant Down syndrome risk RAC-1A80G polymorphic genotype and DS in the Iraqi children sample (A/A Vs. G/G, OR= 1.13,95%C =0.48-2.68 ) (A/A Vs. A/G, OR= 1.2, 95%C = 0.66-2.26 )(G/G vs. A/G, OR=1.08, 95%C = 0.48-2.43 ) respectively. Significant decrease in the concentrations of Hcy and folate in the serum of DS children 5.54 ± 0.94 and 6.99 ± 1.16, then in the control group 7.14 ± 1.46, 7.86 ± 1.78, respectively. We did not detect a significant difference between male and female DS subjects. There was no correlation between the Hcy concentration and folate level of the DS group. The results showed that the frequency of RFC1 alleles and A80G genotypes (GG Vs AA, AAVs AG, GG Vs AG) had no risk with down syndrome in a sample of Iraqi children. Thus, we need further studies with a large sample of children in comparison with mothers of DS birth