Abstract: Microtubules represent one of the major cytoskeletal filament systems of all eukaryotic cells. They play a key role in spatial arrangement of the organelles, intracellular transport, nuclear and cell division, and ciliar motility. Ciliates are ideal model organisms for studying the functional diversity of tubulins. Here, a total of 20 tubulin genes were identified in the macronuclear genome of the ciliate Euplotes octocarinatus. Based on ortholog comparisons and phylogenetic analysis, these genes were clustered into six groups: α-, β-, γ-, δ-, ε- and η-tubulins. Sequence analysis and western blots suggested that the η-tubulin gene of E. octocarinatus required a +1 programmed ribosomal frameshifting to produce complete protein product. The slippery sequence is AAATAAT. We further systematically identified tubulin genes from 9 other ciliates and compared them with E. octocarinatus. The α- and β-tubulins of all investigated free-living ciliates are encoded by multigene families, and each tubulin isotype may be used to form distinct tubulin structures. Phylogenetic analysis showed that α- and β-tubulin genes underwent multiple independent duplications and losses in ciliate. Our study lays a foundation for studying the biological function of tubulins and exploring the mechanisms of microtubule diversity.