The root-rhizosphere interface of Populus is the nexus of a variety of associations between bacteria, fungi, and the host plant and an ideal model for studying interactions between plants and microorganisms. However, such studies have generally been confined to greenhouse and plantation systems. Here we analyze microbial communities from the root endophytic and rhizospheric habitats of Populus deltoides in mature natural trees from both upland and bottomland sites in central Tennessee. Community profiling utilized 454 pyrosequencing with separate primers targeting the V4 region for bacterial 16S rRNA and the D1/D2 region for fungal 28S rRNA genes. Rhizosphere bacteria were dominated by Acidobacteria (31%) and Alphaproteobacteria (30%), whereas most endophytes were from the Gammaproteobacteria (54%) as well as Alphaproteobacteria (23%). A single Pseudomonas-like operational taxonomic unit (OTU) accounted for 34% of endophytic bacterial sequences. Endophytic bacterial richness was also highly variable and 10-fold lower than in rhizosphere samples originating from the same roots. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Pezizomycotina (40%), while the Agaricomycotina were more abundant in the rhizosphere (34%) than endosphere (17%). Both fungal and bacterial rhizosphere samples were highly clustered compared to the more variable endophyte samples in a UniFrac principal coordinates analysis, regardless of upland or bottomland site origin. Hierarchical clustering of OTU relative abundance patterns also showed that the most abundant bacterial and fungal OTUs tended to be dominant in either the endophyte or rhizosphere samples but not both.Together, these findings demonstrate that root endophytic communities are distinct assemblages rather than opportunistic subsets of the rhizosphere.Populus is considered the model organism for the study of woody perennials (46) and represents the first tree genome to be fully sequenced (47). Populus has also received attention in bioenergy research for the production of cellulose-derived biofuels (47). Populus can be grown on land not suitable for food production and increase carbon sequestration, thus minimizing the competition between food and fuel production and reducing the carbon debt incurred through land use changes (39). Populus may also provide an ideal model for understanding a variety of plant-microbe interactions (45). Populus and other members of the Salicaceae are capable of establishing associations with both arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi (14) that may result in unique interactions between these fungi, as well as other endophytic and rhizospheric organisms and the host. Populus bacterial rhizosphere and endophytic constituents have received some attention due to their potential role in phytoremediation of industrial chemicals (33) and heavy metals (4), as well as plant growth-promoting bacteria (PGPB), which benefit plants by providing fixed nitrogen and/or aiding resistance to infection by pathogens ...