Dihydrofolate reductase (DHFR) is a key enzyme in the tetrahydrofolate pathway. This enzyme catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate; a key step in the synthesis of purines, deoxythymidine and methionine. The DHFR structural motif is known to be highly conserved over a wide range of organisms, and the inhibition of DHFR blocks the synthesis of DNA; leading to the cell death. These factors make DHFR a favorable therapeutic target for treating cancer and a wide range of prokaryotic infections. A structure-based alignment was generated using 350 chromosomal and plasmid homology models along with 7 DHFR x-ray crystal structures. Analyses were performed to compare the degree of active site and non-active site conservation in terms of amino acid residues, secondary structure and amino acid residue class. Analyses were also performed to compare chromosomal Bacilli DHFR with plasmid-encoded DHFR to determine the relative degree of conservation. The results of this study will be useful in understanding the likelihood of future evolution of bacterial DHFR in response to selection pressures such those caused by folate therapeutics.