BACKGROUND: Prunus tomentosa Thunb, a fruit tree native to China, is an important and excellent plant material. It exhibits high adaptability to almost all soil types and climatic conditions. The germplasm resources are found in the six geographical regions of China, covering a vast expanse, and comprise a variety of ecological types. However, little is known about genetic diversity and population structure, with its abundant genetic information. Nuclear simple sequence repeat markers, with biparentally inherited characteristics, is powerful for evaluating the genetic diversity of population. Therefore, this research assessed the genetic diversity and population structure of P. tomentosa by collecting extensive samples. The finding will contribute to not only proposing scientific conservation strategies of P. tomentosa but also developing the germplasm to breed novel cherry cultivar. RESULTS: A total of 822 germplasms from 40 populations were used for assessing. The indicators of genetic diversity showed high values, including observable heterozygosity (0.546), expected heterozygosity (0.819), Shannon's information index (I = 1.951), polymorphism information content (0.799). The populations displaying the greatest diversity-Gan-XlM, Shaan-HM, and Gan-ZhC. In addition, extensive genetic differentiation was detected. The genetic differentiation coefficient (F CONCLUSIONS: The P. tomentosa populations showed high or moderate polymorphism. The genetic differentiation within each population was higher than that between populations. There was a significant correlation between the genetic distance and the geographical distance. Based on genetic diversity and population differentiation levers, the most diverse populations-Gan-XlM, Shaan-HS, and Gan-PL from Gansu and Shaanxi provinces, should first be protected and applied to the innovation of cherry germplasm, followed by significantly differentiated populations, including Chuan-Aba, Ning-HlM, and Gan-PL.