The root of the eukaryote tree of life defines some of the most fundamental relationships among species. It is also critical for defining the last eukaryote common ancestor (LECA), the shared heritage of all extant species. The unikont-bikont root has been the reigning paradigm for eukaryotes for more than 10 years 1 but is becoming increasingly controversial 2–4. We developed a carefully vetted data set, consisting of 37 nuclear-encoded proteins of close bacterial ancestry (euBacs) and their closest bacterial relatives, augmented by deep sequencing of the Acrasis kona (Heterolobosea, Discoba) transcriptome. Phylogenetic analysis of these data produces a highly robust, fully resolved global phylogeny of eukaryotes. The tree sorts all examined eukaryotes into three megagroups and identifies the Discoba, and potentially its parent taxon Excavata 5, as the sister group to the bulk of known eukaryote diversity, the proposed Neozoa (Amorphea + Stramenopila+Alveolata+Rhizaria+Plantae SARP 6). All major alternative hypotheses are rejected with as little as ∼50% of the data, and this resolution is unaffected by the presence of fast-evolving alignment positions or distant outgroup sequences. This “neozoan-excavate” root revises hypotheses of early eukaryote evolution and highlights the importance of the poorly studied Discoba for understanding the evolution of eukaryotic diversity and basic cellular processes. Display omitted •A new 37-protein data set produces a robust and rooted eukaryote tree•All examined eukaryotes form three megagroups: Amorphea, SARP, and Excavata•A new neozoan-excavate root is proposed with strong statistical support•Discoba (Excavata) has a long, unique, and largely unknown evolutionary history He et al. developed a novel data set of 37 eukaryotic proteins of bacterial ancestry (euBacs) to explore the major radiations of eukaryotes. A neozoan-excavate root is found, with the little-known Discoba (and potentially its parent taxon Excavata) identified as sister group to the bulk of known eukaryote diversity.