The wild strains of a cultivated species constitute an important breeding material for the genetic enhancement of commercial lines. Cultivated strains of button mushroom, Agaricus bisporus have narrow genetic base due to their origin from few high-yielding hybrids and single spore progenies through tissue culture propagation. The genetic identities of wild and cultivated strains of A. bisporus were established by DNA sequencing of internal transcribed spacer (ITS) regions of 5.8S rRNA gene. Molecular variation among the different strains was assessed using RAPD and AFLP markers. ITS1, 5.8S rRNA gene and ITS2 were 290, 154 and 208 bases, respectively in length in all the strains of A. bisporus studied. SNPs in ITS2 region were identified, which distinguished different strains within the species. RAPD and AFLP markers differentiated all the germplasm strains. White pileus cultivated strains were separated from the wild brown strains by a phylogenetic branch. The wild strains possessed a broad range of genetic variation (53.4%) and exhibited 53.7% genetic distance from the cultivated strains and hybrids, indicating a high level of DNA polymorphism in the germplasm. However, the cultivated strains showed less DNA polymorphism (16.8% genetic variation) as compared to the hybrid cultivars (26.8% variation). The cultivated strains showed a genetic distance of 33.6% from the hybrids and were clearly separated in the UPGMA dendrogram and PCO plot. These results have practical implications for the future breeding programmes as these molecularly diverse germplasm strains could be selected for genetic improvement of this commercially important button mushroom in India.