Crucial to revealing mechanistic details of protein folding is a characterization of the transition state ensemble and its structural dynamics. To probe the transition state of ubiquitin thermal unfolding, we examine unfolding dynamics and kinetics of wild-type and mutant ubiquitin using time-resolved nonlinear infrared spectroscopy after a nanosecond temperature jump. We observe spectral changes on two different time scales. A fast nonexponential microsecond phase is attributed to downhill unfolding from the transition state region, which is induced by a shift of the barrier due to the rapid temperature change. Slow millisecond changes arise from thermally activated folding and unfolding kinetics. Mutants that stabilize or destabilize beta strands III-V lead to a decreased or increased amplitude of the microsecond phase, indicating that the disruption or weakening of these strands occurs in the transition state. Unfolding features from microseconds to milliseconds can be explained by temperature-dependent changes of a two-dimensional free energy surface constructed by the native contacts between beta strands of the protein. In addition, the results support the possibility of an intermediate state in thermal unfolding.