Congenital heart defects are present in 40% to 60% of people with Down syndrome. The 5- methyltetrahydrofolate-homocysteine methyltransferase reductase enzyme (MTRR) is crucial for the regulation of L-homocysteine metabolism. Research shows that single-nucleotide polymorphisms and the environment can affect the development of congenital heart defects in people with Down syndrome. Aim: To determine whether MTRR 66A>G single-nucleotide polymorphism is linked with the occurrence of congenital heart defects in people with Down syndrome. In addition, the aim is to examine the impact of endogenic factors, such as age, nutritional status, and dietary folate intake, as well as periconceptional folic acid intake, smoking, and alcohol consumption, in mothers with Down syndrome children on the development of congenital heart defects. Subjects and Methods: The study includes 155 people with Down syndrome and 148 of their mothers. The control group consists of people with Down syndrome without congenital heart defects. The genotyping of MTRR 66A>G single-nucleotide polymorphism is by the PCRRFLP procedure. Results: The distribution and frequency of genotypes and alleles of MTRR 66A>G singlenucleotide polymorphism are not statistically significantly different in people with Down syndrome and their mothers, i.e. between the group with congenital heart defects and the group without congenital heart defects. Mothers of Down syndrome children who smoked prior to pregnancy and during the data collection are more likely to have a Down syndrome child with ventriculo-coronary arterial connection (p = < 0.001) than mothers who have never smoked (p = 0.024). Mothers of Down syndrome children who ate a Mediterranean diet during pregnancy were statistically more likely to have Down syndrome children with endocardial cushion defect than those who ate a different diet (p = 0.043). Mothers of Down syndrome children who ate a Mediterranean diet during pregnancy had an approximately sixfold increase in the chances of having a child with endocardial cushion defect than those who ate a different diet during pregnancy (OR = 0.163, 95% CI [0.028 – 0.939]. Mothers of Down syndrome children who took periconceptional folic acid supplementation from week four before pregnancy until week eight of pregnancy were more likely to have a Down syndrome child with a congenital heart defect (p = 0.013). Likewise, mothers of Down syndrome children who took periconceptional folic acid supplementation from week four before pregnancy until week eight of pregnancy were more likely to have a Down syndrome child with a atrial septal defect (p = 0.004). Younger mothers of Down syndrome children were more likely to have a Down syndrome child with endocardial cushion defect (p = 0.010). Mothers of Down syndrome children who took periconceptional folic acid supplementation during pregnancy were almost two times more likely to have a Down syndrome child with a congenital heart defect than mothers of Down syndrome children who did not take periconceptional folic acid supplementation during pregnancy (OR = 1.822, 95% CI [1.063 - 3.333]). Likewise, mothers of Down syndrome children with the G allele of MTRR 66A>G single-nucleotide polymorphism had about a ninefold decrease in the chances of having a Down syndrome child with a congenital heart defect if they ate a continental diet during pregnancy (OR = 0.109, 95% CI 95% [0.014 – 0.872]). Moreover, mothers of Down syndrome children with a homozygous genotype mutation of MTRR 66A>G single-nucleotide polymorphism who took periconceptional folic acid supplementation from week four before pregnancy until week eight of pregnancy were more likely to have children with a ventricular septal defect (p = 0.036). Conclusion: The study results show that the mother’s MTRR 66A>G single-nucleotide polymorphism in combination with lifestyle habits such as smoking, diet type, and periconceptional folic acid supplementation may pose a risk for the development of congenital heart defects in children with Down syndrome.