Aim: This study aimed to determine whether polymorphisms of MTHFR C677T/A1298C and MTRR A66G genes, individually or in combination, represented risk factors for the nondisjunction of chromosome 21 in mothers of children with Down Syndrome (DS), considering age, meiotic division, and the numbers and locations of recombinations. Patients and methods: The study included 116 families; 76 were three-member families (mother, father, and child with DS), and 40 were two-member families (mother and child with DS). All DS cases (N = 116) exhibited full trisomy 21. A control group of mothers (N = 141) and fathers (N = 101) consisted of individuals that had a healthy child and no personal or family history of trisomy 21. Subjects and controls were age-matched. PCR genotyping with short tandem repeats was used to determine the parental origin of trisomy 21, meiotic division stage, and the numbers and locations of recombinations. PCR with restriction fragment length polymorphisms was performed to detect MTHFR C677T/A1298C and MTRR A66G polymorphisms. The frequencies of alleles and genotypes were compared between groups with the  2 and Fisher's exact tests. Odds ratios and 95% confidence intervals were calculated to evaluate the allele/genotype risk for trisomy 21. The Spearman rank correlation test was used to test whether individual polymorphisms were associated with chromosome nondisjunctions in meiotic MI and MII divisions or with the numbers and locations of recombinations. Statistical significance was set at P < 0.05. Results: The parental origin of DS was successfully determined in 102 (80%) out of 116 families. Maternal origin of trisomy 21 was found in 93% of cases and was caused by chromosome nondisjunction in the MI (86%) more often than in the MII (14%) division. Paternal origin was found in 5% of cases; mitotic origin occurred in 2% of cases. Mothers of children with trisomy 21 of maternal origin were older than mothers of children with DS of paternal and/or mitotic origin (P = 0.010). In trisomy 21 of maternal origin that resulted from chromosome nondisjunction in the MI division, no recombinations occurred in 79%, one recombination occurred in 18%, and two recombinations occurred in 3% of cases. In cases with a single recombination event, recombination was most commonly located in the distal part of the chromosome (60%). In trisomy 21 caused by chromosome nondisjunction in the MII division, a pericentromeric recombination location was established in all cases with a single recombination event. The frequency distributions of the numbers and locations of recombinations in MI and MII divisions were not significantly different among the different maternal age groups (young ≤36; older 36). No significant differences were found between case and control groups in the frequency of alleles, genotypes, or genotype combinations of the MTHFR C677T/A1298C and MTRR A66G polymorphisms. Additionally, no significant differences were found in the frequency of alleles, genotypes, or genotype combination in case group of mothers according to maternal age, meiotic division, or the numbers and locations of recombinations. Fathers of children with DS showed significantly lower frequencies of the MTHFR 1298CC genotype compared to the control group of fathers (P = 0.029). The frequency distribution of other genotypes and alleles with MTHFR C677T/A1298C and MTRR A66G polymorphisms was not statistically significantly different between the fathers of children with DS and the control group of fathers. Conclusion: Our findings showed that the MTHFR C677T/A1298C and MTRR A66G polymorphisms, individually or in combination, did not represent risk factors for maternal trisomy 21. Although we confirmed that altered recombination was a risk factor for improper separation of chromosomes during meiosis, we did not find an association with the presence of the investigated polymorphisms.