The optimization of industrial mushroom production is dependent on improving the culture process and breeding new strains with higher yields and productivities. There have been few systematic studies on genetic breeding of Agaricus bisporus strain. Hybridization is considered to be one of the effective methods to combine characteristics from different strains and create variability in the existing germ plasm. In the present study, 500 single spore isolates were raised from Agaricus bisporus strain U3 and A15, of which sixteen single spore isolates (seven from A15 and nine from U3) were identified as homokaryons for crossing purposes. Only five crosses were found to be compatible, out of one hundred thirty-eight (138) crosses, and put into fruiting test for confirmation of hybrids. Among these, Hybrid Hb-2 produced maximum yield (22.2 kg/100 kg compost), with healthier morphological traits as compared to parent strain U3 and A15 which produces 18.10 kg/100 kg and 9.50 kg/100 kg compost, respectively, within six weeks harvesting period. RAPD studies showed polymorphism in hybrids and maximum number of bands was produced by OPO-02 in different hybrids. The NTYSc 2.1 software was used to generate a dendrogram based on 73 alleles amplified by RAPD markers, which clustered total hybrids into two main groups in which Ia comprises four isolates viz ., Hb-1, Hb-3, Hb-4, Hb-5 whereas Ib comprise single isolate Hb-2. In all total of five hybrids were differentiated into five variants at 73 per cent similarity coefficient.
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