The genus Lolium L. (Poaceae) is usually regarded as significant forage grass distributed around the world. The origin center for this genus is the Mediterranean region. L. perenne and L. multiflorum (Out-breeding species) are two considerable species, highly grown as crops, fodder, and turf grasses in the world. The relationships and genetic variability among five Lolium species were analyzed utilizing inter simple sequence repeat (ISSR), start codon targeted (SCoT), and internal transcribed spacer (ITS) markers for crop improvement as well as evaluating the differentiation power of ISSR and SCoT molecular markers. This research indicated the productivity of ISSR and SCoT molecular markers to assess the genetic variation and structure among Lolium species. Based on nrITS, ISSR and SCoT markers, clear differentiation was found between outbreeding and inbreeding species. Analyzing the molecular variance (AMOVA) made considerable differences among the species for both markers. STRUCTURE assays showed that the genus Lolium species are genetically differentiated, but have some levels of shared common alleles. All three outcrossing species (L. rigidum, L. multiflorum and L. perenne) are closely associated and introgressions among them but appear to be isolated. These findings will be beneficial for the species conservation and effectively apply the species of Lolium germplasm for crop enhancement and breeding.

属黑麦草属（禾本科）通常被认为是分布在世界各地显著饲草。该属的起源中心是地中海地区。L. perenne和L. multiflorum（外育种）是两个重要的物种，在世界上作为农作物、饲料和草坪草高度生长。五种黑麦草间的亲缘关系及遗传变异利用简单序列重复序列间 (ISSR)、起始密码子靶向 (SCoT) 和内部转录间隔区 (ITS) 标记对作物进行了分析，并评估了 ISSR 和 SCoT 分子标记的分化能力。该研究表明ISSR和SCOT分子标记的生产力，以评估之间的遗传变异和结构黑麦草品种。基于 nrITS、ISSR 和 SCoT 标记，发现远交和近交物种之间存在明显差异。分析分子方差 (AMOVA) 使两种标记的物种之间存在相当大的差异。结构分析表明，黑麦草属物种是遗传分化的，但具有某些水平的共享等位基因。所有三个异交物种（L.rigidum , L. multiflorum和L. perenne ) 密切相关，它们之间有基因渗入，但似乎是孤立的。这些发现将有利于物种保护，有效利用黑麦草种质资源进行作物增产育种。