Due to unsatisfying attempts to fingerprint Auricularia polytricha, two different molecular maker systems-Inter-Simple Sequence Repeats (ISSR) and Sequence-related amplified polymorphism (SRAP)-were established and tested to quantify molecular diversity among 19 strains of this fungus. A total of 202 (99.0%) and 459 (95.9%) polymorphic bands were detected by 13 ISSR primers and 14 SRAP primer combinations, respectively. By parsimony method, a phylogenetic tree was constructed based on each analysis; the two trees show that 19 A. polytricha strains were distributed into five or four groups. These results demonstrated that both methods were suitable for discriminating among strains of A. polytricha, and the novel SRAP markers are more efficient and preferable. The result also indicated the high level of genetic diversity of A. polytricha and their relationship between each other. These findings would benefit future research in A. polytricha, especially in breeding and medicine development. It also gives a useful method for fingerprinting of other fungi.