Abstract: In order to investigate the genetic differentiation, we studied the genetic structure and genetic variation of 23 Chinese indigenous horse breeds and one thoroughbred horse population using 25 microsatellite markers. The number of alleles, polymorphism information content (PIC) and heterozygosity showed that genetic diversity in Chinese horses was higher than the thoroughbred horse. Neighbour-joining (NJ) dendrogram clustered Chinese horse populations and the thoroughbred horse into different groups. Furthermore, Chinese horse populations were grouped into several different phylogenetic clusters which corresponded to the geographic regions. Cluster analysis was performed by the Multivariate Statistical Package (MVSP), demonstrating that thoroughbred horse was discriminated from all the studied populations based on the three-dimensional scatter plot for the first three factors. Two-dimensional scatter plot for the first two princi-pal factors divided Chinese horse populations into five groups: southern China, Tibet, Xinjiang and Qinghai, Inner Mongolia, and Northeast China. Genetic structure revealed by the software package Structure 2.2 dis-played five potential elementary genetic groups in Chinese modern horses.