Mytilaria laosensis is a fast-growing tropical broadleaf tree that is extensively used for wood production and has significant ecological benefits. To investigate the genetic diversity and population structure of M. laosensis, eight major natural populations in China were analyzed by using simple sequence repeat (SSR) markers. A total of 88 microsatellite-containing fragments were obtained by the method of magnetic bead enrichment, among which 26 pairs of SSR primers were screened out and used to generate a total of 190 alleles among 152 individuals. The average of observed number of alleles, Shannon’s information index and polymorphism information content per locus were 18.3, 1.1577 and 0.7759, respectively, implying a high level of genetic diversity in M. laosensis populations. The variation within populations accounted for 81.74 % of total variation based on analysis of molecular variance. Cluster analysis divided the eight populations into four groups, among which five populations from the southern parts of Guangxi province were classified as one major group. Mantel test showed that there was highly significant correlation between Euclidean genetic distance and geographic distance, suggesting that geographic isolation contribute to the high genetic diversity of M. laosensis. Together, these could provide support for the feasibility of exploration and utilization of M. laosensis in subtropical areas of East Asia including Jiangxi, Hunan and Fujian province of China.