Ammopiptanthus nanus(M. Pop.) Cheng f. is a very important resource plant that integrates soil and water conservation, afforestation of barren mountains, and ornamental, medicinal, and scientific research functions and is also a critically endangered plant in China, remaining in only six small fragmented populations in the wild. These populations have been suffering from severe anthropomorphic disturbances, causing further losses in genetic diversity. However, its genetic diversity level and genetic differentiation degree among the fragmented populations are still unclear. Inthis study, DNA was extracted from fresh leaves from the remnant populations ofA. nanus, and the inter-simple-sequence repeat (ISSR) molecular marker system was used to assess its level of genetic diversity and differentiation. The result was that its genetic diversity is low at both species and population levels, with only 51.70% and 26.84% polymorphic loci, respectively. The Akeqi population had the highest genetic diversity, whereas the Ohsalur and Xiaoerbulak populations had the lowest. There was significant genetic differentiation among the populations, and the value of the genetic differentiation coefficient (Gst) was as high as 0.73, while the gene flow value was as low as 0.19 owing to spatial fragmentation and a serious genetic exchange barrier among the populations. It is suggested that a nature reserve and germplasm banks should be established as soon as possible for elimination of the anthropomorphic disturbances, and mutual introductions between the populations and introduced patches of the species, such as with habitat corridors or stepping stones, should be performed simultaneously to improve the genetic diversity of the isolated populations for the conservation of this plant.