We report the reinvestigation of the high-resolution rotational spectrum of estradiol. After removing the known spectral lines corresponding to three conformers of estradiol identified in the gas phase before, a large number of spectral lines remained unassigned in the spectrum. The observation of remaining lines is a common feature in spectra obtained by broadband rotational spectroscopy. In our reinvestigation, the detection of certain patterns resulted in two new sets of experimental rotational constants. Here we describe a systematic analysis, which together with quantum-chemical computations culminated in the assignment of two estrone conformers, namely exhibiting the trans- and the cis-arrangement of the hydroxy group attached to the rigid steroid backbone. Estrone and estradiol only differ in two atomic mass units, and they show a dynamic interconversion equilibrium under certain conditions, which might also have been the case in our experiments due to the heating temperature of 195 °C. The results illustrate the potential of high-resolution rotational spectroscopy to discern between structurally related molecules and to provide their gas-phase structures without information beforehand exploiting the benefit of having remaining unassigned rotational transitions in the spectrum.