Agaricus bisporus is a delicious, nutritious mushroom with medical function. Cross breeding is limited due to its taxonomic contradiction. We analyzed rDNA ITS sequences of two high quality Agaricus bisporus strains (No.Tb07, As2796). Tb07 strain was selected from foreign mushroom by CSUFT under protection. As2796 strain was a high quality local strain in nature in China. By sequencing the ITS regions and comparing with those sequences from Genebank by BLAST search, the results concluded that the ITS region length of Tb07 strain was 701 bp and As2796 strain 695 bp. GC content ranged from 41.72% to 42.22% in ITS1 region and 43.89% to 44.31% in ITS2 region. ITS1 and ITS2 sequences of two strains had seven bp differences. Tb07 strain had 314 bp in ITS1 and 262 bp in ITS2, As2796 strain had 315 bp in ITS1 and 255 bp in ITS2. It suggested that in natural population of Agaricus bisporus had a little variation. From ITS sequence differences, we can select two parents which have a distance relationship for cross breeding. It was important to explore its wild germ plasm resource and increased its genetic variation using cross breeding with aid of molecular data.