Abstract: In this study, the 5 SrDNA,45 SrDNA and repeated sequences including pAs 1 and pSc 119.2 were used as probes to carry out in situ hybridization with chromosomes of wild Hordeum brevisubulatum,and karyotype of wild H.brevisubulatum chromosomes was analyzed by observing the position of the probe on chromosome and the strength of marking signal.The results showed that 5 SrDNA was located on the short arms of chromosome（chr） 10 and chr 14,while 45 SrDNA was located on the short arms of chr 6,chr 10,chr 11,chr 12,chr 13 and chr 14.In addition, most of the fluorescence signal of pAs 1 DNA appeared at the end of chromosomes, and some appeared in the centromere region.Meanwhile, the fluorescent signal of pSc 119.2 appeared at the tip of the short arms of chr 7 and chr 14,and also at the tip of a short arm of chr 10.There was no fluorescent signal of pSc 119.2 in other chromosome because of tip missing.The karyotype formula was 2 n=4 x=28=28 m（12 SAT） and the karyotype belonged to type 1 A.The asymmetry coefficient of karyotype was 57.227%.