Eukaryotic cells create internal order by using protein motors to transport molecules and organelles along cytoskeletal tracks. Recent genomic and functional studies suggest that five cargo-carrying motors emerged in primitive eukaryotes and have been widely used throughout evolution. The complexity of these "Toolbox" motors expanded in higher eukaryotes through gene duplication, alternative splicing, and the addition of associated subunits, which enabled new cargoes to be transported. Remarkably, fungi, parasites, plants, and animals have distinct subsets of Toolbox motors in their genomes, suggesting an underlying diversity of strategies for intracellular transport.