Isoprene (2-methyl-1,3-butadiene) is highly abundant in the atmosphere, second only to methane in hydrocarbon emissions. In contrast to the most stable trans rotamer, structural characterization of gauche-isoprene has proven challenging: it is weakly polar, present at the level of only a few percent at room temperature, and structurally complex due to both torsional and methyl tunneling motions. gauche-Isoprene has been observed by two distinct but complementary experimental approaches: chirped-pulse Fourier transform microwave (CP-FTMW) spectroscopy coupled with cryogenic buffer gas cooling, and cavity-enhanced FTMW spectroscopy with a pulsed discharge source. Thermal enhancement of the gauche population (from 1.7% to 10.3%) was observed in the cryogenic buffer gas cell when the sample was preheated from 300 to 450 K, demonstrating that high-energy rotamers can be efficiently isolated under our experimental conditions. Rotational parameters for the inversion states (0+/0-) have been determined for the first time, aided by calculations at increasing levels of theoretical sophistication. From this combined analysis, the inversion splitting Δ E and the Fbc Coriolis coupling constant between the two inversion states have been derived.