Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.