Eukaryotic cells physically manipulate their environments; swimming through liquids, crawling across surfaces, and actively ingesting objects large and small. Inside these cells lies a seething mass of cytoplasm through which thousands of different objects are pushed and pulled to specific cellular locations. These and other dynamic processes are controlled by polymer systems called the cytoskeleton. The two most common cytoskeletal polymers—actin and microtubules—evolved over a billion years ago and are still used today by animals, plants, and their unicellular relatives. Although the proteins that comprise actin and microtubule polymers themselves are surprisingly similar across species, the hundreds of different proteins that regulate their dynamics are wildly variable, contributing to aspects of organismal diversity critical to human health and agriculture. We combine cell biology, comparative genomics, and phylogenetics to understand the evolution, diversification, and regulation of actin and microtubule networks. Here we harness the burgeoning wealth of fully sequenced genomes and molecular tool development to (1) identify the molecular mechanisms that specify distinct cytoskeletal functions, and (2) determine how and when the cytoskeleton diversified during eukaryotic evolution.