Proteins have dynamic structures that undergo chain motions on time scales spanning from picoseconds to seconds. Resolving the resultant conformational heterogeneity is essential for gaining accurate insight into fundamental mechanistic aspects of the protein folding reaction. The use of high-resolution structural probes, sensitive to population distributions, has begun to enable the resolution of site-specific conformational heterogeneity at different stages of the folding reaction. Different states populated during protein folding, including the unfolded state, collapsed intermediate states, and even the native state, are found to possess significant conformational heterogeneity. Heterogeneity in protein folding and unfolding reactions originates from the reduced cooperativity of various kinds of physicochemical interactions between various structural elements of a protein, and between a protein and solvent. Heterogeneity may arise because of functional or evolutionary constraints. Conformational substates within the unfolded state and the collapsed intermediates that exchange at rates slower than the subsequent folding steps give rise to heterogeneity on the protein folding pathways. Multiple folding pathways are likely to represent distinct sequences of structure formation. Insight into the nature of the energy barriers separating different conformational states populated during (un)folding can also be obtained by resolving heterogeneity.