The thyroid in vertebrates and its homolog, the endostyle in nonvertebrate chordates, share a molecular code for dorsoventral patterning. Little is yet known, however, about mechanisms that pattern the endostyle's anterior–posterior (AP) axis. To extend our understanding of thyroid development and evolution, we studied Oikopleura dioica, a larvacean urochordate that retains a chordate body plan as adults. Transcription factor expression domains revealed AP regionalization of the endostyle, with expression of Otx rostrally, Hox1 caudally, and two Pax2/5/8 paralogs centrally. Comparative analysis suggested that the endostyle of stem chordates expressed orthologs of these genes and that ancestral subfunctions partitioned differentially among lineages. Because the ordered expression of Otx, Pax2/5/8, and Hox1 displays patterning in both the endodermally derived endostyle and the ectodermally derived central nervous system, we propose that this gene set belonged to the developmental genetic toolkit of stem bilaterians and repeatedly provided AP positional information in various developmental situations. Developmental Dynamics 237:1490-1499, 2008. © 2008 Wiley-Liss, Inc.