Abstract: In order to accurately analysis genetic similarity of Flammulina velutipes germplasm resources, ISSR, RAPD and SRAP molecular markers were used to differentiate 6 Flammulina velutipes strains. A dendrogram, constructed using similarity coefficients ranging from 0.49 to 0.85 clustered the 6 strains into 3 groups at a similarity coefficient of 0.59. One of them consisted of 8903, another Jin-21, and the third group the remainders. Our data demonstrated that the simultaneous use of 3 different types of markers based on different principles represented a powerful tool for analyzing genetic relationships among Flammulina velutipes strains. This method is advantageous to the identification and evaluation of Flammulina velutipes germplasm resources.