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Abstract Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox geneTlxin Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization ofTlxexpression indicate an upregulation ofTlxduring medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoanPodocoryna carneaand the scyphozoanPelagia noctiluca. These results suggest thatTlxplays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.