The morphological origin of anisotropic charge transport in uniaxially strain aligned poly(3-hexylthiophene) (P3HT) films is investigated. The macroscale field effect mobility anisotropy is measured in an organic thin film transistor (OTFT) configuration and compared to the local aggregate P3HT mobility anisotropy determined using time-resolved microwave conductivity (TRMC) measurements. The field effect mobility anisotropy in highly aligned P3HT films is substantially higher than the local mobility anisotropy in the aggregate P3HT. This difference is attributed to preferentially aligned polymer tie-chains at grain boundaries that contribute to macroscale charge transport anisotropy but not the local anisotropy. The formation of sharp grains between oriented crystalline P3HT, through tie chain removal by thermal annealing the strained aligned films, results in an order of magnitude drop in the measured field effect mobility for charge transport parallel to the strain direction. The field effect mobility anisotropy is cut in half while the local mobility anisotropy remains relatively constant. The local mobility anisotropy is found to be surprisingly low in the aligned films, suggesting that the π−π stacking direction supports charge carrier mobility on the same order of magnitude as that in the intrachain direction, possibly due to poor intrachain mobility through chain torsion.