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 to 26.5 GHz and 26.5 to 40.0 GHz. The spectra of all singly-substituted heavy atom isotopologues 13C, 18O and 34S in their natural abundances could be measured and assigned to determine the gas-phase substitution rs and semi-experimental reSE structures of the most abundant conformer. The spectrum of the 33S isotopologue with its nuclear quadrupole coupling hyperfine structure was analysed, yielding the complete quadrupole tensor with high accuracy. 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 kJ mol−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 33S nucleus are discussed within the frame of the current literature. This work provides an important contribution to the study and characterisation of sulfur-containing volatile organic compounds.