Poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene), PBTTT, is a semiconducting polymer that forms thin film transistors (TFTs) with high field effect mobility on silicon dioxide dielectrics that are treated with alkyltrichlorosilanes (∼0.2 to 0.5 cm2/V s) but forms TFTs with poor mobility on bare silicon dioxide (<0.005 cm2/V s). The microstructure of spin-coated thin films of PBTTT on these surfaces was studied using synchrotron X-ray diffraction and atomic force microscopy. PBTTT crystallizes with lamellae of π-stacked polymer chains on both surfaces. The crystalline domains are well-oriented relative to the substrate in the as-spun state and become highly oriented and more ordered with thermal annealing in the liquid crystalline mesophase. Although the X-ray scattering from PBTTT is nearly identical on both surfaces, atomic force microscopy showed that the domain size of the crystalline regions depends on the substrate surface. These results suggest that electrical transport in PBTTT films is strongly affected by the domain size of the crystalline regions and the disordered regions between them.