We examined the development of olfactory neuropils in the hemimetabolous insect Locusta migratoria with an emphasis on the mushroom bodies, protocerebral integration centers implicated in memory formation. Using a marker of the cyclic adenosine monophosphate (cAMP) signaling cascade and lipophilic dye labeling, we obtained new insights into mushroom body organization by resolving previously unrecognized accessory lobelets arising from Class III Kenyon cells. We utilized antibodies against axonal guidance cues, such as the cell surface glycoproteins Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I), as embryonic markers to compile a comprehensive atlas of mushroom body development. During embryogenesis, all neuropils of the olfactory pathway transiently expressed Sema 1a. The immunoreactivity was particularly strong in developing mushroom bodies. During late embryonic stages, Sema 1a expression in the mushroom bodies became restricted to a subset of Kenyon cells in the core region of the peduncle. Sema 1a was differentially sorted to the Kenyon cell axons and absent in the dendrites. In contrast to Drosophila, locust mushroom bodies and antennal lobes expressed Fas I, but not Fas II. While Fas I immunoreactivity was widely distributed in the midbrain during embryogenesis, labeling persisted into adulthood only in the mushroom bodies and antennal lobes. Kenyon cells proliferated throughout the larval stages. Their neurites retained the embryonic expression pattern of Sema 1a and Fas I, suggesting a role for these molecules in developmental mushroom body plasticity. Our study serves as an initial step toward functional analyses of Sema 1a and Fas I expression during locust mushroom body formation. J. Comp. Neurol. 520:2021–2040, 2012. © 2011 Wiley Periodicals, Inc.