Abstract Background Both euarthropods and vertebrates have tripartite brains. Several orthologous genes are expressed in similar regionalized patterns during brain development in both vertebrates and euarthropods. These similarities have been used to support direct homology of the tripartite brains of vertebrates and euarthropods. If the tripartite brains of vertebrates and euarthropods are homologous, then one would expect other taxa to share this structure. More generally, examination of other taxa can help in tracing the evolutionary history of brain structures. Tardigrades are an interesting lineage on which to test this hypothesis because they are closely related to euarthropods, and whether they have a tripartite brain or unipartite brain has recently been a focus of debate. Results We tested this hypothesis by analyzing the expression patterns of six3, orthodenticle, pax6, unplugged, and pax2/5/8 during brain development in the tardigrade Hypsibius exemplaris—formerly misidentified as Hypsibius dujardini. These genes were expressed in a staggered anteroposterior order in H. exemplaris, similar to what has been reported for mice and flies. However, only six3, orthodenticle, and pax6 were expressed in the developing brain. Unplugged was expressed broadly throughout the trunk and posterior head, before the appearance of the nervous system. Pax2/5/8 was expressed in the developing central and peripheral nervous system in the trunk. Conclusion Our results buttress the conclusion of our previous study of Hox genes—that the brain of tardigrades is only homologous to the protocerebrum of euarthropods. They support a model based on fossil evidence that the last common ancestor of tardigrades and euarthropods possessed a unipartite brain. Our results are inconsistent with the hypothesis that the tripartite brain of euarthropods is directly homologous to the tripartite brain of vertebrates.