There has been intense interest in understanding the self-assembled structure of conductive polymer nanowires due to the potential application in photovoltaics area. In the present study, free-standing poly(3-hexylthiophene) (P3HT) thin films consisting of H-aggregated and recently reported J-aggregated nanowires (NWs) were respectively prepared by the vacuum filtration method. The microstructure differences of both samples were studied in detail by combining the multiple techniques, i.e., synchrotron radiation WAXD, solid-state NMR, and IR spectroscopy. It is found that there is stronger pi-pi interaction ascribed to the slightly closer interchain stacking in J-aggregates than that in H-aggregates, although both aggregates take the same form I crystal modification. The subtle structural difference in the interchain stacking distance (Delta d(020) = 0.04 angstrom) is further manifested in the thermal behavior of both aggregates as indicated by the DSC curves, in which J-aggregates exhibit much higher melting point than that of H-aggregates (Delta T = 14 degrees C). Meanwhile, in situ synchrotron radiation WAXD study suggests that the low-temperature thermal transitions in both aggregates are associated with the shrinkage of the interchain distance. It is expected that this work will be helpful in understanding the structure properties relationship of varying P3HT aggregate types.