Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host. The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.