The microstructural evolution of martensitic AISI 52100 steel during non-isothermal tempering under a series of constant heating rates was investigated through differential scanning calorimetry and dilatometry. The kinetic parameters for different tempering stages during heating were studied using the differential isoconversional method and also the Kissinger method as comparison. It is found that the activation energies obtained by both methods coincided with the literature. The activation energies obtained by the Kissinger method were constant, while those obtained by the isoconversional method varied with the fraction converted and presented respective variation trends. The kinetics of phase transformation during isothermal tempering was then simulated based on the Johnson-Mehl-Avrami-Kolmogorov (JMAK) theory. The calculated isothermal kinetics coincided with the experimental data from the literature. The Vickers hardness of samples with the same fraction converted under non-isothermal tempering at 0.33 K s(-1) and isothermal tempering at both 373 and 473 K was measured to verify the applicability of non-isothermal to isothermal transformation. Increasing the isothermal tempering temperature accelerated the reduction of hardness when compared with non-isothermal tempering. Mechanisms for the differences in hardness between these two types of tempering were discussed.