Abstract Aim To explore the genetic and phylogeographic structure of a temperate forest species, Pinus strobiformis Englem., in a subtropical region in the context of climate change during the Pleistocene. It is expected that the colder conditions during glacial stages favoured range expansions of P. strobiformis, thus promoting gene flow. Location Mexico and the United States. Methods Estimates of genetic diversity and structure were obtained using chloroplast microsatellite loci of 23 populations of P. strobiformis across its entire range, seven neighbouring populations of Pinus ayacahuite Ehrenb. ex. Schtdl, and one population of Pinus flexilis James. Results The genetic diversity of P. strobiformis (He = 0.856) was found to be high, especially in western populations, whereas eastern populations were less variable and more genetically similar to P. ayacahuite of central Mexico. We found evidence of significant phylogeographic structure (NST = 0.444; P =0.026), high genetic structure (RST = 0.270), and isolation by distance. Pairwise RST andsamova(spatial analysis of molecular variance) results indicated an east–west partition of genetic variation, with populations within each group showing little differentiation and no isolation by distance. Main conclusions The phylogeographic structure of P. strobiformis across the entire range was pronounced, with two main genetic and geographic groups separated by the Chihuahuan Desert. However, within each of the two groups there was little population differentiation and no isolation by distance, suggesting genetic connectivity as a result of population expansions within these areas during glacial stages.