Structure-property relationships are of fundamental importance to develop quantitative models describing charge transport in organic semiconductor based electronic devices, which are among the best candidates for future portable and lightweight electronic applications. While microstructural investigations, such as those based on X-rays, electron microscopy, or polarized optical probes, provide necessary information for the rationalization of transport in macromolecular solids, a general model predicting how charge accommodates within structural maps is not yet available. Therefore, techniques capable of directly monitoring how charge is distributed when injected into a polymer film and how it correlates to structural domains can help fill this gap. Supported by density functional theory calculations, here we show that polarized charge modulation microscopy (p-CMM) can unambiguously and selectively map the orientational order of the only conjugated segments that are probed by mobile charge in the few nanometer thick accumulation layer of a high-mobility polymer-based field-effect transistor . Depending on the specific solvent-induced microstructure within the accumulation layer, we show that p-CMM can image charge-probed domains that extend from submicrometer to tens of micrometers size, with markedly different degrees of alignment. Wider and more ordered p-CMM domains are associated with improved carrier mobility, as extracted from device characteristics. This observation evidences the unprecedented opportunity to correlate, directly in a working device, electronic properties with structural information on those conjugated segments involved in charge transport at the buried semiconductor-dielectric interface of a field-effect device.