The microwave spectra of thiophene and 2-acetylthiophene were recorded in the frequency range from 2 to 40 GHz using two molecular jet Fourier transform microwave spectrometers. For 2-acetylthiophene, two conformers with a syn and an anti orientation of the S1–C2 and C6O bonds (with respect to the C2–C6 bond) were identified, and the syn-conformer was more stable. The spectra of the ³⁴S- and ¹³C-isotopologues of syn-2-acetylthiophene were also assigned, and the semiexperimental equilibrium structure could be determined. Compared to thiophene, at the substitution position, the S1–C2 and C2C3 bond lengths both increase by about 0.007 Å, and the bond angle S1–C2C3 decreases by 0.06°, noticeably larger than the experimental uncertainties. A–E torsional splittings were observed due to internal rotation of the methyl group hindered by a barrier height of 330.187(35) and 295.957(17) cm⁻¹ for the syn-conformer and the anti-conformer, respectively. Geometry and internal rotation parameters are compared with those of related thiophene derivatives, as well as those of furan and 2-acetylthiophene to gain a better understanding of structure determination in the presence of methyl internal rotation.