Agaricus bisporus, commonly known as the button mushroom, is the most widely cultivated species of edible fungi. The cultivars used by growers over the world are suspected to come from the same restricted pool of strains, and the genetic base of all the present day hybrids is very narrow. The aim of this study was to assess the genetic variability among traditional and modern commercially used A. bisporus strains. Fourteen codominant microsatellite markers ( AbSSR) were used to characterize 75 cultivated genotypes from European spawn makers, maintained in the Collection of Agaricus in Bordeaux (CGAB) since 1990. To our knowledge, it is the most extensive sample ever studied. Seven main groups were identified which corresponded to the six ancestral lineages and the hybrids belonging to either U1 or U3 sub-group of strains. Thirty-three U1-like cultivars could not be differentiated. Very few strains have a distinct and typical SSRs pattern. Based on our results, we proposed also a cultivar identification key with a limited number of markers in order to optimize forthcoming SSRs fingerprinting. For three hybrids that seemed to be genetically identical to Horst-U1 at heterokaryotic level, the analysis of each constituting nuclei has demonstrated allelic rearrangement, suggesting essentially derived varieties. The efficiency of microsatellite markers and implications of these results for germplasm management, breeding strategy and variety identification are discussed.