In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the pK(a) of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the pK(a) of the NS position of the bound substrate. We observe a gradual increase in NS basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 pK(a) of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.