A novel and major cannabinoid (epicannabidiol hydrate) present in hemp plants and oils was isolated and characterized by a combination of flash chromatography and liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS). This novel cannabinoid differs from cannabidiol (CBD) by the absence of a double bond in the terpene ring and the presence of an additional hydroxyl group in the same terpene ring. The isolation procedure involved the use of normal phase chromatography with a silica gel preparative column, followed by reversed phase chromatography with a C18 preparative column. In this way, other major cannabinoids present in the samples, such as cannabidiol and Δ9-tetrahydrocannabinol, were separated and the focus was placed on the novel cannabinoid compound. Exact accurate masses were obtained for the compound of interest at m/z = 333.2424 in positive ion mode and m/z = 331.2279 in negative ion mode. Additional MS-MS analysis in negative ion mode revealed the position of the additional hydroxyl group in the molecule. Finally, the structural characterization was corroborated with 1H-NMR and 13C-NMR analysis, thus verifying the exact chemical structure of this novel cannabinoid, which has not previously been reported in hemp samples.