At the onset of neural development, pioneer growth cones can migrate over epithelia or neuroepithelia along stereotyped routes that establish the pattern of initial neural tracts. These migration routes may reflect the arrangement of distinct epithelial or neuroepithelial domains. In grasshopper limb buds, a pair of afferent pioneer neurons arise in the tibia and their growth cones migrate on a stereotyped path through the limb to the CNS. In the limb buds, circumferentially-oriented epithelial domains expressing semaphorin-I, annulin, or alkaline-phosphatase, and a longitudinal domain, expressing engrailed, have been described. Using multiple-labeling techniques, we describe the relationships of these domains to each other and to the pioneer neuron pathway. Taken together, these domains establish an orthogonal pattern of regionally specific epithelial molecular markers. During much of their migration across the limb epithelium, the pioneer growth cones are in register with the axes of circumferential or longitudinal epithelial domains.