Abstract: A total of 112 primer pairs were successfully designed， of which 97 primers produced stable and specific bands in 39 Agaricus bisporus genotypes （including 31 single spore isolates and 8 other strains）. Twenty five primer pairs showed polymorphism among single spore isolates and 19 primer pairs followed the law of segregation in single spore isolates population. Further study showed that 67 alleles were detected with the average of 268.The average of Neis genetic diversity index （H） was 0.502 3. The genetic similarity coefficient ranged from 0.336 3 to 0.864 1 with an average of 0538 5. The UPGMA cluster analysis based on SSR data showed that these parental lines could be classified into two groups at the threshold of 0.46， which was related to their pedigree analysis. The results indicated that molecular markers was usable in map structure， analysis of genetic polymorphism in A.bisporus. It also provided the genetic basis for the breeding of new varieties of A.bisporus or the collection and management of germplasm resources.

Key words: Agaricus bisporus, single spore isolates, SSR, polymorphism, genetic diversity