Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan-tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.