The bifunctional a-amylase/subtilisin inhibitor (BASI) encoded by the lsa gene on barley chromosome 2HL could inhibit wheat a-amylase activity, thereby reducing sprouting and improving the quality of wheat.In our work, Regenerated plants were derived from immature embryo culture of hybrids of common wheat varieties Zhengmai 9023, CB037 and Zhongmai 16 with the wheat-barley 2H alien substituion lines 2H(A) and 2H(B) after callus induction, subculture and differentiation.The presence of barley 2H chromatin was detected in regenerated plants (SC1) and their selfing progenies (SC2-5) using SSR molecular markers of the barley second homology group and further more were identified in selected SC4 and SC5 lines using genomic in situ hybridization that barley genomic DNA as probe and CS genomic DNA for blocking.The lsa gene from the identified SC4 and SC5 lines was also amplified by lsa specific primers.Eventually, we identified lines with barley 2HL chromosome including a homozygous translocation line, heterozygotic translocation lines, monotelosomic substitution lines, double telocentric substitution lines, and an isochromosome substitution line carring the lsa gene.The chromosome configurations of the homozygous translocation line 32-4-11 and two heterozygotic translocation lines 32-4-9 and 32-4-18 in meiosis were also analyzed.