Abstract: In order to study the feasibility of ISSR and SSR markers in the identification of new lines of hybrid Hordeum brevisubulatum, 12 ISSR primers and 6 pairs of SSR primer were used to identify the fingerprints of the two new lines. The results showed that the average percentage of polymorphic loci of ISSR labeled primers and SSR labeled primers were 77.93% and 69.22%, respectively, both of which exhibited high polymorphism; The genetic similarity coefficient and cluster analysis of the two markers showed that the new hybrid lines Y₃₃ and P₁₃ were biased to the recurrent parent Hordeum brevisubulatum; The combination of two different fragments labeled with different primers could distinguish the materials. The fingerprints of ISSR and SSR markers could objectively and truly detect the genetic differences between parents and hybrid offspring, which could be used for the identification of distant hybrids of Hordeum brevisubulatum and provide basis for the approval of new varieties.