The genetic constitution of fifteen materials derived from the cross wheat (Triticum aestivum L. cv. 'Chinese Spring') X barley (Hordeum vulgare L. cv. 'Betzes') was analyzed, and six disomic alien substitution lines were screened by GISH. The chromosome configurations in pollen mother cells at meiotic metaphase I (PMCs M I) of F, from each disomic substitution line respectively crossed with double ditelocentric lines 2A, 2B and 2D of 'Chinese Spring' were observed, and a set of wheat-barley disomic alien substitution lines 2H(A), 2H(B) and 2H(D) were obtained. The RFLP analysis with the probe psr131 on the short arm of wheat homeologous group 2 combining with four restriction enzymes were carried out. The results indicated that the probe psr131 could be used as molecular marker to tag the barley chromosome 2H. The barley chromosome 2H had good genetic compensation ability for wheat chromosomes 2B and 2D in vitality and other agronomic characters. The result of testing seed was that the wheat appearance starch quality had been changed from the half-farinaceous of 'Chinese Spring' to the half-cutin of substitution lines by transferring the barley chromosome 2H to wheat.