Pteroceltis tatarinowii, a relic tree endemic to China, is mainly distributed in limestone mountains and has a wide geographical range. In this study, 12 microsatellite primer pairs were assayed to analyse the genetic pattern and gene flow among 461 individuals sampled from 23 wild populations of P. tatarinowii. A high level of genetic diversity was detected based on high values of total alleles (159), the number of alleles (NA = 6.373), expected heterozygosity (HE = 0.696) and observed heterozygosity (HO = 0.679). The high genetic diversity in this species was attributed to its long-life history, large-scale geographical distribution and wind dispersal breeding system. Low genetic differentiation (GST = 0.137, FST = 0.138) was found among the populations of P. tatarinowii. The genetic variation occurred mostly within the populations. Gene flow was estimated to be 1.562. This moderate level of gene flow could decrease interpopulation differentiation by buffering against genetic drift and improving gene exchange. The spatial genetic structure of P. tatarinowii occurred at the whole-region scale (r = 0.311, P < 0.05) and in the region of South China (r = 0.453, P < 0.05), which might be related to terrain heterogeneity and the demographic history of P. tatarinowii. The distinct east-west high mountains distributed in South China might serve as physical barriers to seed and pollen flow. The isolation and local adaptation of different refugia could further limit normal gene flow. In addition, far-apart populations might fail to effectively disperse pollen and seeds. Based on the above-mentioned results, some tentative suggestions for protection are presented for this species.