Here we describe the developmental expression of the three iroquois genes ( BfIrxA, BfIrxB, and BfIrxC) of amphioxus. BfIrxB transcription is first detected at the gastrula stage in mesendoderm just within the dorsal lip of the blastopore (a probable homolog of Spemann’s organizer) and in ectoderm. In early neurulae, expression begins in presumptive pharyngeal endoderm, somitic mesoderm, and neural plate. Mid-neurulae express BfIrxB throughout the hindbrain, posterior somites, pharyngeal endoderm, and notochord. In early larvae, expression is largely downregulated in the nerve cord, somites and notochord, but remains strong in the pharyngeal endoderm associated with the forming gill slits; also, a late expression domain appears in the ciliary tuft ectoderm. BfIrxA and BfIrxC, are not as widely expressed as BfIrxB. Both are first expressed in the presumptive hindbrain and presumptive pharyngeal endoderm at the early neurula stages. In the mid-neurula, additional expression domains appear in the extremities of the notochord. Neural expression is downregulated by late neurula. In the early larva, expression is chiefly limited to pharyngeal endoderm associated with the forming gill slits, excepting a small new domain of BfIrxC (not BfIrxA) expression in the ciliary tuft ectoderm. In comparison to developing vertebrates, embryos and larvae of amphioxus express iroquois genes in fewer tissues. Thus, iroquois genes of the proximate ancestor of the vertebrates evidently assumed numerous new roles during vertebrate evolution, including the division of the central nervous system into several sub-regions along its anteroposterior axis.