Monolayer-thickness two-dimensional layers of α,ω-dihexylsexithiophene (α,ω-DH6T) exhibit field-effect hole mobility of up to 0.032 cm2 V−1 s−1, higher than previously reported for monolayers of other small-molecule organic semiconductors. In situ measurements during deposition show that the source-drain current saturates rapidly after the percolation of monolayer-high islands, indicating that the electrical properties of α,ω-DH6T transistors are largely determined by the first molecular monolayer. The α,ω-DH6T monolayer consists of crystalline islands in which the long axes of molecules are oriented approximately perpendicular to the plane of the substrate surface. In-plane lattice constants measured using synchrotron grazing-incidence diffraction are larger in monolayer-thickness films than the in-plane lattice constants of several-monolayer films and of previously reported thick-film structures. Near-edge X-ray absorption fine structure spectroscopy (NEXAFS) reveals that the larger in-plane lattice constant of single-monolayer films arises from a larger tilt of the molecular axis away from the surface normal. NEXAFS spectra at the C 1s and S 2p edges are consistent with a high degree of molecular alignment and with the local symmetry imposed by the thiophene ring. The high mobility of holes in α,ω-DH6T monolayers can be attributed to the reduction of hole scattering associated with the isolation of the thiophene core from the interface by terminal hexyl chains.