The genus Pseudoroegneria (Nevski) Löve (Triticeae, Poaceae) with St genome accounts for more than 60% of the perennial Triticeae speciation, and provide abundant germplasm for forage crop and pasture breeding. The diploid species Pseudoroegneria libanotica (2n = 14) possess the most ancient St genome. The homoeology between Pse. libanotica and other Triticeae species chromosomes has not been investigated. Hence, characterization of the St genome is critical for a better understanding of the evolution of Triticeae species. We distinguished seven homoelogous groups of Pse. libanotica by mapping 14 single copy gene probes derived from wheat and developed three new tandem repeats to distinguish non-homologous chromosomes of Pse. libanotica. Based on FISH data, the molecular karyotype of Pse. libanotica was established. Compared with other Triticeae species, most of the homoelogous chromosomes were relatively conserved, except pericentric inversion in 7St, and duplicated fragments in 2St_S from 5St_L were obtained. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of Pse. libanotica in crop breeding.