Abstract The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport and cytokinesis, and is responsible for diverse eukaryotic morphologies. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton we provide the first single cell transcriptomes from uncultivable, free-living unicellular eukaryotes: the radiolarian species Lithomelissa setosa and Sticholonche zanclea . Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these to a revised phylogeny of Rhizaria reveals lineage-specific gene duplications and neo-functionalization of α and β tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution of uncultivable protist species.