Ganoderma (lingzhi) is a famous herbal medicine and edible supplement in oriental countries for a long history. In this study, a simple micellar electrokinetic chromatography (MEKC) method was established for the analysis of nucleosides and bases, the major bioactive components in Ganoderma for the first time. By optimizing the borate concentration, the sodium dodecyl sulfate (SDS) concentration and the pH value of running buffer, 10 nucleosides and bases achieved an ideal separation. In real sample analysis, the developed method was successfully used to determine the 10 target analytes in 23 batches of Ganoderma samples from different regions. Results indicated that contents of 10 investigated analytes in each sample showed obvious variation. The principal components analysis (PCA) and hierarchical cluster analysis (HCA) analysis classified the samples into three groups, and the HCA tree visualized the relationships which was mainly contributed by geographical partition. The results indicated geographical origin to be an important factor that affect the accumulation of nucleosides and bases in Ganoderma. In summary, this study provides a simple and practical strategy for quality assessment and cultivation reference of Ganoderma.