The macro-evolutionary consequences of recent climate change remain controversial and there is little paleobotanical or morphological evidence that Pleistocene (1.8-0.12 Ma) glacial cycles acted as drivers of speciation, especially among lineages with long generation times, such as trees. We combined genetic and ecogeographic data from two closely related North American tree species, Populus balsamifera and P. trichocarpa (Salicacaeae) to determine if their divergence coincided with and was possibly caused by Pleistocene climatic events. We analyzed 32 nuclear loci from individuals of P. balsamifera and P. trichocarpa to produce coalescent-based estimates of the divergence time between the two species. We coupled the coalescent analyses with paleodistribution models to assess the influence of climate change on species’ range. Further, measures of niche overlap were used to investigate patterns of ecological differentiation between species. We estimated the divergence date of P. balsamifera and P. trichocarpa at approximately 75 Ka, during the middle of the Wisconsin glaciation (115–12 Ka). Significance tests of niche overlap, in conjunction with genetic estimates of migration, suggested that speciation occurred in allopatry, possibly resulting from the environmental effects of Pleistocene glacial cycles. Our results indicate the divergence of keystone tree species, that have shaped community diversity in northern North American ecosystems, was recent and may have been a consequence of Pleistocene-era glaciation and climate change.