A repeat sequence producing the multiple hybridization signals on chromosomes was obtained by constructing a Cot-1 DNA library and screening with a technique of fluorescence in situ hybridization (FISH) in Leymus secalinus (Georgi) Tzvel. Further cloning and sequence analysis revealed that the sequence is tandem repeated in the genome with a repeated motif of 90 bp. The repetitive sequence was named as pLs-90. Karyotyping with pLs-90 revealed that each chromosome of L. secalinus could be well identified by the different FISH patterns with multiple hybridizations on centromeric, subcentromeric, interstitial, or subtelomeric ?regions. PLs-90 can not only be an idea chromosome maker for Leymus germplasm identification and utilization but also a powerful tool for studying the genome evolution of different species in Triticeae.