The Brown-eared pheasant (Corossoptilon mantchuricum) is a critically endangered endemic to China, listed in the national first-class protected bird in China. At present, the distribution area of the Brown-eared pheasant was isolated by Yellow River and Taihang Mountains to form the three regions and the three isolated populations. Those isolated geographic populations are the middle population located in Lvliang Mountains, Shanxi; the east population in Dongling Mountains, Beijing and Xiaowutai Mountains, Hebei and the west population in Huanglong Mountains, Shaanxi, among which Shanxi is the distribution center and abundance center of the Brown-eared pheasant. So far the most researches concerning the Brown-eared pheasant mainly focus on biological, ecological, resource investigation and protection. Using molecular biological method to the research of genetic diversity of the Brown-eared pheasant is still at the initial stage. For the protection of germplasm resources of the Brown-eared pheasant and to optimize the genetic structure of captive populations and further reintroduction, we detected the genetic relationship of 35 individuals in two populations in Shanxi from Pangquangou Nature Reserve and Taiyuan Zoo were studied by using ISSR markers. Ten pairs of primers with repeatable clear and stable bands from 20 pairs of ISSR primers for 35 samples of the two populations were selected, and 65 DNA bands were amplified with polymerase chain reactions, 77% of which were polymorphic bands. PhylTools software was used to calculate Nei's genetic distance and genetic similarity of each individual from the two populations. The analysis from Nei's genetic distance and genetic similarity of individuals from each other showed that the means of genetic similarity of individuals from the two populations were 0.5061 and 0.7591, and the means of Nei's genetic distance were 0.4939 and 0.2409, respectively. As a whole, the means of Nei's genetic distance and genetic similarity of all 35 individuals are 0.3723 and 0.6310, respectively, which indicated that the genetic variation of individuals in the two populations were at low level, with relatively close genetic distance. We also performed cluster analysis using group average clustering method with (0, 1) matrix and Nei's genetic distance of the 35 individuals. The results showed that the samples with close genetic distance had close genetic relationship. Most individuals from the same population were grouped together; it indicated that those from similar ecological and geographical environment had the closer relationship. The individuals from Pangquangou Nature Reserve had lower genetic similarity and further genetic distance with each other than the individuals from Taiyuan Zoo, which indicated that those from Pangquangou Nature Reserve had further genetic relationship than those from Taiyuan Zoo. This may due to the free-ranging method and the annual supplement of picking up wild eggs and hatching in Pangquangou Nature Reserve. With this approach, we can easily identify genetic relationship of individuals and select those with relatively far genetic distance for high quality germplasm resource. This method would serve as a novel tool in the research of endangered birds.