The genus Populus comprises some of the most commercially exploited, pioneer forest trees distributed throughout the northern-hemisphere. The high level of morphological diversity, extensive inter-species hybridization, and low level of DNA sequence variation among species in this group have impeded the progress of taxonomic and phylogenetic studies. We used nuclear genomic data based upon inter simple sequence repeat (ISSR) variability, a highly variable class of molecular markers to determine the genetic relationships among species of the genus Populus. Species of the section Populus (Leuce) clustered together suggesting monophyly of the section Populus. The Eurasian members of section Populus (P. alba, P. davidiana, and P. tremula) showed closer genetic relationships to each other than to two North American aspens (P. tremuloides and P. grandidentata) of the same section. In contrast to previous phylogenetic studies, P. nigra showed a close genetic relationship to species of the section Tacamahaca. This relationship is in agreement with various phenotypic traits, interfertility and chemistry of bud exudates and serves as evidence for introgression between P. nigra and species of the section Tacamahaca. Overall, the genetic relatedness estimates based on nuclear ISSR data were congruent with phylogenetic trees based on other molecular (RFLP and DNA sequence) and morphological data, but provided better resolution in assessing the genetic relatedness among closely related taxa, and provided genetic evidence for previously suspected introgressions.