The chelicerate body plan is distinguished from other arthropod groups by its division of segments into 2 tagmata: the anterior prosoma (“cephalothorax”) and the posterior opisthosoma (“abdomen”). Little is understood about the genetic mechanisms that establish the prosomal-opisthosomal (PO) boundary. To discover these mechanisms, we created high-quality genomic resources for the large-bodied spider Aphonopelma hentzi. We sequenced specific territories along the antero-posterior axis of developing embryos and applied differential gene expression analyses to identify putative regulators of regional identity. After bioinformatic screening for candidate genes that were consistently highly expressed in only 1 tagma (either the prosoma or the opisthosoma), we validated the function of highly ranked candidates in the tractable spider model Parasteatoda tepidariorum. Here, we show that an arthropod homolog of the Iroquois complex of homeobox genes is required for proper formation of the boundary between arachnid tagmata. The function of this homolog had not been previously characterized, because it was lost in the common ancestor of Pancrustacea, precluding its investigation in well-studied insect model organisms. Knockdown of the spider copy of this gene, which we designate as waist-less, in P. tepidariorum resulted in embryos with defects in the PO boundary, incurring discontinuous spider germ bands. We show that waist-less is required for proper specification of the segments that span the prosoma-opisthosoma boundary, which in adult spiders corresponds to the narrowed pedicel. Our results demonstrate the requirement of an ancient, taxon-restricted paralog for the establishment of the tagmatic boundary that defines Chelicerata.