Conjugated polymers usually form crystallized and amorphous regions in the solid state simultaneously, making it difficult to accurately determine their precise microstructures. The lack of multiscale microstructures of conjugated polymers limits the fundamental understanding of the structure–property relationships in polymer‐based optoelectronic devices. Here, crystals of two typical conjugated polymers based on four‐fluorinated benzodifurandione‐based oligo(p‐phenylene vinylene) (F4BDOPV) and naphthalenediimide (NDI) motifs, respectively, are obtained by a controlled self‐assembly process. The strong diffractivity of the polymer crystals brings an opportunity to determine the crystal structures by combining X‐ray techniques and molecular simulations. The precise polymer packing structures are useful as initial models to evaluate the charge transport properties in the ordered and disordered phases. Compared to the spin‐coated thin films, the highly oriented polymer chains in crystals endow higher mobilities with a lower hopping energy barrier. Microwire crystal transistors of F4BDOPV‐ and NDI‐based polymers exhibit high electron mobilities of up to 5.58 and 2.56 cm2 V−1 s−1, respectively, which are among the highest values in polymer crystals. This work presents a simple method to obtain polymer crystals and their precise microstructures, promoting a deep understanding of molecular packing and charge transport for conjugated polymers. Conjugated polymer microwire crystals are obtained from solvated aggregates. The precise crystal packing and electronic structure in the polymer microwires are evaluated for understanding of the charge transport properties. Polymer crystal transistors of F4BDOPV‐2T exhibit higher electron mobilities of up to 5.58 cm2 V−1 s−1 with a much lower hopping energy barrier compared with conventional thin‐film transistors.