[OBJECTIVE] The objectives of this study were to use Roche 454 GS FLX system to develop SSR markers for Armillaria luteo-virens. These datasets will be valuable for detecting genetic diversity and population structure of this species. [METHODS] We collected Armillaria luteo-virens samples from Yushu in Qinghai province, China. Total RNA was isolated by using the TRIzol reagent, after that we constructed cDNA library and performed one quarter plate of the whole run 454 pyrosequencing. We selected 98 primer pairs randomly from the 321 SSRs to evaluate their application and the polymorphism across 66 individuals (Armillaria luteo-virens) representing 3 wild populations. [RESULTS] Roche 454 sequencing yielded 197,121 reads with a total nucleotide size of 88,585,965bp. 27 of 98 SSRs loci were polymorphic. Numbers of alleles (Na) ranged from 2 to 8. Expected heterozygosity (HE) ranged from less than 0. 001 to 0. 810 at locus ALV65, while observed heterozygosity (Ho) from 0 at loci AIV64 and AIV92 to 0. 900 at loci ALV8. We found no evidence of linkage disequiliburium, however 10 of 27 SSR markers showed significant deviation from Hardy-weinberg equilibrium. [CONCLUSION] These remaining 17 pairs of Armillaria luteo-virens SSR markers will be valuable for future research on detecting population structure and conservation of this species.