Clathrate hydrates (CHs) are believed to exist in cold regions of space, such as comets and icy moons. While spectroscopic studies have explored their formation under similar laboratory conditions, direct structural characterization using diffraction techniques has remained elusive. We present the first electron diffraction study of tetrahydrofuran (THF) and 1,3-dioxolane (DIOX) CHs in the form of nanometer-thin ice films under an ultrahigh vacuum at cryogenic temperatures. Using reflection high-energy electron diffraction, we show that THF CH grows readily on various substrates during thermal annealing of an amorphous ice mixture of THF and water, and the formation is independent of the nature of the substrate. The growth of DIOX CHs on a Au(111) substrate is similar. A comparison of electron diffraction patterns with calculated X-ray diffraction patterns indicates that THF and DIOX form structure II CH (51264) with a lattice constant of ∼17.2 Å (cubic Fd3̅m). Both CHs were also grown on Ru(0001) and were examined by reflection absorption infrared spectroscopy. A direct comparison of diffraction data to infrared spectra as a function of the temperature further demonstrates the strength of multiple probes in examining complex systems possessing diverse molecular interactions.