In organic thin film transistors (OTFT), modifying the molecular chemical structure affects the molecular packing and thin film morphology, which both sensitively influence the charge carrier mobility. A detailed understanding of the interplay of molecular chemical structure, molecular packing, and thin film morphology is therefore necessary to improve OTFT performance. Fluorene-bithiophene-fluorene (FTTF) derivatives have demonstrated great potential for use as the active layer for OTFTs. A series of FTTF asymmetrically substituted derivatives were synthesized to fine-tune the film properties. In this study, the thin film microstructure details of FTTF and several FTTF derivatives with asymmetrically substituted alkyl-chains of different lengths are studied and compared with their electrical performance in thin film transistor devices. The respective unit cells were characterized using grazing incidence X-ray diffraction (GLXD), and for FTTF the detailed molecular packing was determined from the GIXD intensities in combination with packing simulation. The alignment of the FTTF core in the thin film phase was further characterized using near edge X-ray absorption fine structure (NEXAFS) spectroscopy for films prepared under different processing conditions and under different film growth stages. For well-packed asymmetric FTTF derivatives, we find that the conjugated FTTF core is in direct contact with the substrate surface, and its single alkyl chain is aligned away from that interface. The impact of the detailed microstructure in the films on the charge mobility is discussed.