In the present study, the genetic diversity of 69 Ganoderma species from various regions was determined by different molecular markers, including the internal transcribed spacer (ITS) rRNA, a partial β-tubulin gene, and mitochondrial small-subunit ribosomal (SSU) rDNA gene as well as randomly amplified polymorphic DNA (RAPD) analysis. The size of the ITS rDNA regions and mitochondrial SSU rDNA gene from different Ganoderma species varied from 625 to 673 bp and 656 to 2,040bp, respectively, and those of the partial β-tubulin gene sequence were 419 bp. Phylogenetic analysis based on the ITS region, the partial β-tubulin gene, and the mitochondrial SSU rDNA gene reveal that Ganoderma species are largely divided into two groups. Interestingly, most of the Ganoderma lucidum strains could be classified into 1 group, while other Ganoderma species divided into several groups (4 or 5 groups) in phylogenetic tree. One fragment unique to G. lucidum was selected from the RAPD profile and then sequenced. One primer pair (designated as KGS-F and KGS-R) based on this specific fragment was designed to amplify a 559 bp DNA fragment within the sequenced region. A single band with the expected size of 559 bp was observed from G. lucidum, except for G. lucidum strains from China, Canada, and Taiwan. This specific marker for G. lucidum from RAPD analysis, also supported by the phylogenetic analysis of the ITS, partial β-tubulin gene, and mitochondrial SSU rDNA sequences, will be useful for the PCR-based identification of G. lucidum in research applications as well as in the market.