Isothermal nucleation of gas hydrates in two-layer water–organic liquid systems was studied with the use of Sapphire Rocking Cell RCS6 with six transparent sapphire cells by thermal analysis under gas pressure. The experiments were conducted at constant supercooling (about 20.2 °C). Decane (both with and without surfactants Surfynol 420 and Span 80) and crude oils were used as organic liquids. It was observed that gas hydrate nucleation occurred at water–stainless steel–gas and water–sapphire–organic liquid three-phase contact lines rather than at water–gas or water–organic liquid interfaces. The empirical survival curves were constructed for the entire set of data. Two series of experiments for the system of water–decane–Surfynol 420–methane with the same sample were also conducted. The series of experiments differed with cell angle relative to the horizontal (45° and 14°). Comparison of the observed thermal effects with data of visual examination for this system allowed nucleation rates of methane hydrate on the different nucleation sites to be determined.