In order to develop a simple, efficient, and sensitive method for comprehensive analysis of the nucleosides and nucleobases in natural products, a zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) method for the simultaneous determination of 16 nucleosides and nucleobases has been studied. A mechanistic study confirmed that ZIC-HILIC separation showed a mixed-mode effect of both hydrophilic and electrostatic interactions. This method was validated to be precise, accurate, and sensitive with overall precision (intra- and interday) less than 1.8% (RSD), and LOD and LOQ was in the range of 0.005-0.029 μg/mL and 0.018-0.096 μg/mL, respectively. With this method, the nucleosides and nucleobases in Ganoderma of different species (G. atrum, G. lucidum, and G. sinense) and origins were quantified. The results showed that the contents varied with the species and origins. With the aid of hierarchical cluster analysis (HCA), cultivated Ganoderma from different origins and species were successfully discriminated. It is for the first time that the content of nucleosides and nucleobases in G. atrum is reported and compared. Our data showed that HILIC had advantages as a useful and potential tool for the study of the bioactive components in Ganoderma as well as their quality control, and could therefore be used for the determination of the analytes in other natural products.