To relate the bainitic microstructures to the mechanical properties of steel, the average dislocation density needs to be determined. Using X-ray diffraction and diffraction line broadening analysis, this research quantifies the average dislocation density in the four bainite phase matrices, (upper bainite, upper and lower bainite mixture, lower bainite, lower bainite and martensite mixture), which are transformed in a wide range of isothermal temperatures. The effects of isothermal temperatures on the average dislocation density are assessed for different thermal dynamic driving forces in terms of activation energy and cooling rate. It is found that as isothermal holding temperature is increased, the dislocation density in the bainite matrix decreases from 1.55 × 10¹⁷ to 8.33 × 10¹⁵ (m⁻²) due to the reduction in the plastic deformation in the austenite in the transformation. At the same time, the activation energy required decreases only after passing the martensite and lower bainite mixed phase. A new method for better estimating the average dislocation density in bainitic steel is also proposed.