Methylenetetrahydrofolate reductase (MTHFR) family of proteins catalyze the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. They contain a flavin adenine dinucleotide (FAD) as the cofactor and the enzyme in eukaryotes, except in yeast, is known to be allosterically regulated by S-adenosylmethionine. Some cardiovascular diseases, neural tube defects, neuropsychiatric diseases and certain type of cancers in humans are associated with certain polymorphisms of MTHFR. Here, we analyzed 57 of MTHFR polypeptide sequences by multiple sequence alignment and determined previously unrecognized conserved residues that may have a functional or structural importance. A previously unrecognized ATP synthase motif was found in all of the examined plant MTHFRs, suggesting a different functional capability to the plant MTHFRs in addition to the known function. On a phylogenetic tree built, eukaryotic MTHFR proteins formed a clear cluster separated from prokaryotic and archeal relatives. The sequence identities among the eukaryotic MTHFRs were less divergent than the bacterial MTHFRs.