Abstract: Based on the specifical combination between biotin and streptavidin coated magnetic beads, biotinylated probes composed of di-nucleotide repeat were used to isolate microsatellite sequences from genome of Agaricus blazei. DNA digested fragments of Agaricus blazei ligated with artificial adapters in both ends were hybridized with the labeled probes absorbed in magnetic beads. After eluting the unhybridized DNA, the enriched fragments in beads were released to establish the microsatellite library. Of 522 clones detected by PCR with corresponding repeat primers, 48 clones have shown to be positive reaction. After sequencing, 32 clones were proved to contain microsatellite sequences (SSR), with a microsatellite enrichment efficiency of 67% as against positive clones and of 6% as against the total detected clones. By deleting the duplicated and unavailable sequences, 12 microsatellite loci were selected to design primers for identifing 85 candidate mutants of Agaricus blazei induced by Co60 irradiation. The total 17 mutants could be determined by 4 pairs of primer. It demonstrates that some microsatellite loci can be applied to screen and distinguish the mutants of Agaricus blazei induced by Co60 irradiation.