The hindbrain is a vertebrate-specific embryonic structure of the central nervous system formed by iterative transitory units called rhombomeres (r). Rhombomeric cells are segregated by interhombomeric boundaries which are prefigured by sharp gene expression borders. The positioning of the first molecular boundary within the hindbrain (the prospective r4/r5 boundary) responds to the expression of an Iroquois (Irx) gene in the anterior (r4) and the gene vHnf1 at the posterior (r5). However, while Irx3 is expressed anteriorly in amniotes, a novel Irx gene, iro7, acts in teleosts. To assess the evolutionary history of the genes responsible for the positioning of the r4/r5 boundary in vertebrates, we have stepped outside the gnathostomes to investigate these genes in the agnathans Lethenteron japonicum and Petromyzon marinus. We identified one representative of the Hnf1 family in agnathans. Its expression pattern recapitulates that of vHnf1 and Hnf1 in higher vertebrates. Our phylogenetic analysis places this gene basal to gnathostome Hnf1 and vHnf1 genes. We propose that the duplication of an ancestral hnf1 gene present in the common ancestor of agnathans and gnathostomes gave rise to the two genes found in gnathostomes. We have also amplified 3 Irx genes in L. japonicum: LjIrxA, LjIrxC, LjIrxD. The expression pattern of LjIrxA (the agnathan Irx1/3 ortholog) resembles those of Irx3 or iro7 in gnathostomes. We propose that an Irx/hnf1 pair already present in early vertebrates positioned the r4/r5 boundary and that gene duplications occurred in these gene families after the divergence of the agnathans.