Abstract: Genetic diversity of twenty-five populations of Quercus mongolica, which is a dominant tree species from Northeast China, was investigated by the Inter-Simple Sequence Repeat (ISSR) technique, using ten specific and stable primers selected from 60 primers. A total of 71 sites were generated, 56 of which (78.87%) were polymorphic. The data were analyzed by PopGene. The average percentage of polymorphic loci of populations was 45.2% and demonstrated high genetic diversity (the average value of Shannon index (I) was 0.25). To some extent, both gene flow and genetic diversity were present among populations (N_m was 1.3818, average Nei’s index was 0.1068, and average G_st was 0.2657). The gene proportion of diversity reached 73.43% within population but was 26.57% among populations. This demonstrated that genetic variation of Q. mongolica populations was mainly within populations. According to cluster analysis and the law of geographic variation, the populations were classified into two large groups: the Xiaoxinganling group and the Changbaishan group. These results supplement information for protection and utilization of Q.mongolica, and also provide further data for the study of genetic variation and species differentiation of Quercus.