Thickness-dependent crystal structure, surface morphology, surface energy, and molecular structure and microstructure of a series of polycrystalline pentacene films with different film thickness ranging from several monolayers to the several hundred nanometers have been investigated using X-ray diffraction (XRD), atomic force microscopy (AFM), contact angle meter, and Raman spectroscopy. XRD studies indicate that thin film polymorphs transformation behaviours are from the orthorhombic phase to the thin-film phase and then to the triclinic bulk phase as measured by the increased tilt angle (θtilt) of the pentacene molecule from the c-axis toward the a-axis. We propose a growth model that rationalizes the θtilt increased along with increasing film thickness in terms of grain size and surface energy varying with film growth using AFM combined with contact angle measurements. The vibrational characterizations of pentacene molecules in different thickness films were investigated by Raman spectroscopy compared to density functional theory calculations of an isolated molecule. In combination with XRD and AFM the method enables us to distinguish the molecular microstructures in different thin film polymorphs. We proposed a methodology to probe the microscopic parameters determining the carrier transport properties based on Davydov splitting and the characteristics of aromatic C–C stretching modes in Raman spectra. When compared to the triclinic bulk phase at a high thickness, we suggest that the first few monolayer structures located at the dielectric surface could have inferior carrier transport properties due to weak intermolecular interactions, large molecular relaxation energy, and more grain boundaries.