Almost all eukaryote life forms have now been placed within one of five to eight supra-kingdom-level groups using molecular phylogenetics1-4. The `phylum' Hemimastigophora is probably the most distinctive morphologically defined lineage that still awaits such a phylogenetic assignment. First observed in the nineteenth century, hemimastigotes are free-living predatory protists with two rows of flagella and a unique cell architecture5-7; to our knowledge, no molecular sequence data or cultures are currently available for this group. Here we report phylogenomic analyses based on high-coverage, cultivation-independent transcriptomics that place Hemimastigophora outside of all established eukaryote supergroups. They instead comprise an independent supra-kingdom-level lineage that most likely forms a sister clade to the `Diaphoretickes' half of eukaryote diversity (that is, the `stramenopiles, alveolates and Rhizaria' supergroup (Sar), Archaeplastida and Cryptista, as well as other major groups). The previous ranking of Hemimastigophora as a phylum understates the evolutionary distinctiveness of this group, which has considerable importance for investigations into the deep-level evolutionary history of eukaryotic life—ranging from understanding the origins of fundamental cell systems to placing the root of the tree. We have also established the first culture of a hemimastigote (Hemimastix kukwesjijk sp. nov.), which will facilitate future genomic and cell-biological investigations into eukaryote evolution and the last eukaryotic common ancestor.