Roegneria kamoji Ohwi exited widely in China, Japan and Korea. Genetic variations and variations within species of R. kamoji were reported in previous studies. In this study, we using artificial hybridization, morphology comparison, thremmatology and genome analysis studies for exploring the differentiation of seven populations of R. kamoji from Sichuan, Qinghai, Xinjiang, Zhejiang and Shandong. The results showed that: 1) the heights, length of spike, length of lemma were different for these populations. The phenotypic traits of F₁ hybrid were better than their parents, while the floret number and width of glume were inferior to their parents. 2) Meiotic pairing in hybrids averaged 19.77 to 20.91 bivalents per cell, 0.18 to 2.46 univalents per cell, and a low frequency of trivalents and tetravalents. 3) The fertility and natural seed set of the F₁ hybrids ranged from 78.9% to 89.67%, 43.05% to 77.98%, respectively. The lowest seed set (43.05%) was found in hybrid between Zhejiang and Yibin. The above results suggested that differentiations were existed in seven populations of R. kamoji, and it positively correlated with the geographical distribution of the populations. The natural seed set of hybrid between Zhejiang and Yibin was only 43.05%, which is lower than 50%, certifying that reproductive isolation existed between these two populations of R. kamoji.