Knowledge of genetic variation and connectivity is of great importance to protect endangered species. The region of East Sichuan and West Hubei in China is the biodiversity center of broad-leaved trees in the world, which experienced severe forest fragmentation during the past several decades. Magnolia patungensis is an endangered horticultural tree species endemic to this area. For 16 microsatellite markers, low genetic variations (HE = 0.228–0.364) and high genetic differentiation (FST = 34.0%, G″ST = 53.4%) were found in the four extant M. patungensis populations. These were explained by the genetic bottleneck, genetic drift, inbreeding and restricted gene flow. The low level of historical gene flow (Nmhis = 0.762) among M. patungensis populations was attributed to its limited dispersal ability of pollens and seeds, and the complex terrains of habitats (high mountains, deep valleys, and dense forests). Lower contemporary gene flow (Nmcon = 0.474) suggested that the gene flow was further hindered by recent habitat fragmentations. As for conservation strategies, all remaining populations should be protected to maximize gene representation of the tree species; the artificial pollination is recommended within each population in order to increase seed setting.