A rapid, sensitive, and validated method was developed for the simultaneous determination of four iminosugars, 1-deoxynojirimycin (DNJ), d-fagomine (FAG), 2-O-a-d-galactopyranosyl-DNJ (Gal-DNJ), and 4-O-β-d-glucopyranosyl-fagomine (Glu-FAG), in mulberry leaves. The method used hydrophilic interaction chromatography coupled with tandem mass spectrometry. Based on this method, the seasonal variations of iminosugars in the leaves of different mulberry species (Morus alba, Morus multicaulis Perr, Morus atropurpurea Roxb. and Morus wittiorum Hand-Mazz.) collected in Guangzhou, China, during 10 consecutive months in 2013 were investigated. The results indicated that the maximum content of DNJ in the leaves of all four species occurred in summer (June or July). The highest FAG level was found in spring (April or May). The highest levels of Gal-DNJ and Glu-FAG were recorded in autumn (September or October).