A correlation between the hole transport and corresponding structural properties of the bulk regioregular poly(3-hexylthiophene) (rr-P3HT) is studied as a function of temperature by the time-of-flight (TOF) and wide angle X-ray diffraction (WAXD) techniques. The thermally-reversible structural evolution along the (100) and (020) directions in a semi-crystalline rr-P3HT can be divided into two distinct temperature regions. At T > 120 °C, a large thermal expansion along the π–π stacking direction in the nanocrystals and a deteriorated ordering in the material result in negative slopes of temperature and electric field dependences of hole mobility. The WAXD data suggest that the hole transport is limited by a decrease in the crystallinity and by an increase in the hopping distance along the π-π stacking direction, while the Gaussian Disorder Model (GDM) with temperature-independent parameters cannot be applied. At T < 120 °C, the transport-related structural changes are negligible and the temperature and electric field dependences of hole mobility can be described by the GDM with constant energetic (σ ∼ 120 meV) and positional disorder parameters (Σ ∼ 3.33). These values suggest that the hole transport is limited by the amorphous phase, as commonly seen in disordered polymers. Moreover, a regiorandom P3HT (rra-P3HT), which shows a temperature-independent intermolecular distance of ∼15.3Å, provides a route for separate examination of the amorphous phase in rr-P3HT.