Incompatibility of interspecies cross between sweet cherry and Cerasus tomentosa was overcome by successive of hand pollination, and the new germplasm G3 was obtained by using embryo culture technology to overcome the embryo abortion of distance hybridization. The G3 S-genotype was identified in seedling using AS-PCR, the self-incompatibility gene of parent was used as molecular marker to validate the pedigree of hybrid G3, RAPD analysis further demonstrated that G3 share characteristic RAPD fragments of both parents. This result suggested that AS-PCR was an effective and credible method to identify the progeny of interspecies hybrid in Prunus. And the cross could be a promising way to enhance the genetic background. DNA molecular marker realized the identification and pre-selection in the seedling stage. So the developing breeding program would be accelerated by the new way.