AbstractRapid growth of genome data provides opportunities for updating microbial evolutionary relationships, but this is challenged by the discordant evolution of individual genes. Here we build a reference phylogeny of 10,575 evenly-sampled bacterial and archaeal genomes, based on a comprehensive set of 381 markers, using multiple strategies. Our trees indicate remarkably closer evolutionary proximity between Archaea and Bacteria than previous estimates that were limited to fewer “core” genes, such as the ribosomal proteins. The robustness of the results was tested with respect to several variables, including taxon and site sampling, amino acid substitution heterogeneity and saturation, non-vertical evolution, and the impact of exclusion of candidate phyla radiation (CPR) taxa. Our results provide an updated view of domain-level relationships.