We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C > T, 1298A > C, and 1793G > A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing (TM) technology. The MTHFR 677C > T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P < 0.001 for both age groups) in both genders. The effect of MTHFR 1298A > C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C > T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R-2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C > T polymorphism. The common 1298A > C polymorphism had a minor elevating effect on tHcy, whereas the 1793G > A polymorphism had a lowering effect on tHcy.