Thymidylate synthase (TSase) catalyzes a hydride transfer in the last step of the de novo biosynthesis of the DNA nucleotide thymine. We compared two isozymes, namely, TSase from Escherichia coli (ecTSase) and TSase from Bacillus subtilis (bsTSase) that represent a case of divergent evolution. Interestingly, a highly conserved histidine (H147 of ecTSase) was proposed to serve a critical role in catalysis, but in bsTSase it is naturally substituted by valine (Val). Yet, bsTSase is more active than ecTSase, and the intrinsic kinetic isotope effects (KIEs) of both are temperature-independent, suggesting a similarly well-organized transition state (TS) for the catalyzed hydride transfer. To examine the role of that histidine (His) in TSase catalysis, we examined the kinetics of H147V ecTSase, which “bridges” between these two TSases. In contrast to both wild-type TSases, the single mutation results in deficient catalysis. The mutation leads to intrinsic KIEs that are temperature-dependent, indicating a substantial imperfection in its TS. The findings reveal two important features: a direct role of H147 in the hydride transfer step catalyzed by the ecTSase and the evolutionary compensation for its deficiency in bsTSase via extensive polymorphism across the protein. Very different active site residues are observed for these evolutionarily divergent isozymes, which result in a well-organized TS for both. It is suggested that evolutionary pressure compensated for the H to V substitution at the active site of bsTSase by polymorphism leading to a well-organized TS in both enzymes.