We report on the structures of two conformers of 2-thiophenecarboxaldehyde as obtained using a combination of molecular jet Fourier-transform microwave spectroscopy and quantum chemical calculations. The microwave spectrum was recorded using two spectrometers operating in the frequency ranges of 2.0 GHz to 26.5 GHz and 26.5 GHz to 40.0 GHz. The spectra of all singlysubstituted heavy atom isotopologues 13 C, 18 O, and 34 S in their natural abundances could be measured and assigned to determine the experimental gas-phase substitution rs and semi-empirical re SE structures of the most abundant conformer. The spectrum of the 33 S isotopologue with its nuclear quadrupole coupling hyperfine structure was analyzed, yielding the complete quadrupole tensor with aa = 22.63799(76), = bb cc = 18.4892( 14), and ab = 12.002(33) MHz.Quantum chemical calculations for the electric field gradient tensor including corrections with a calibration factor determined from 27 previous studies on the 33 S species predicted the 33 S nuclear quadrupole coupling constants of 2-thiophencarboxaldehyde with high quality. The experimental results are used to map the observed rotational constants to the corresponding molecular structure obtained from quantum chemical calculations, which predicted two conformers with an energy difference of about 6 kJmol 1 at the MP2/6-311++G(d,p) level of theory. Insight into the conformational stability of aromatic heterocyclic carboxaldehydes and bond situations of the sulfur atom extracted from the hyperfine structure of the 33 S nucleus are discussed within the frame of the current literature. This work provides an important contribution to the study and characterization of sulfur-containing volatile organic compounds.