A cluster of three Specificity Protein (Sp) genes (Sp1-4, Sp5 and Sp6-9) is thought to be ancestral in both chordates and the wider Eumetazoa. Sp5 and Sp6-9 gene groups are associated with embryonic growth zones, such as tailbuds, and are both Wnt/β-catenin signalling pathway members and targets. Currently, there are conflicting reports as to the number and identity of Sp genes in the cephalochordates, the sister group to the vertebrates and urochordates. We confirm the SP complement of Branchiostoma belcheri and Branchiostoma lanceolatum, as well as their genomic arrangement, protein domain structure and residue frequency. We assay Sp5 expression in B. lanceolatum embryos, and determine its response to pharmacologically increased β-catenin signalling. Branchiostoma possesses three Sp genes, located on the same genomic scaffold. Phylogenetic and domain structure analyses are consistent with their identification as SP1-4, SP5 and SP6-9, although SP1-4 contains a novel glutamine-rich N-terminal region. SP5 is expressed in axial mesoderm and neurectoderm, and marks the cerebral vesicle and presumptive pharynx. Early exposure to increased β-catenin caused ubiquitous SP5 expression in late gastrula, while later treatment at gastrula stages reduced SP5 expression in the posterior growth zone during axis elongation. Amphioxus possess a typical invertebrate eumetazoan SP complement, and SP5 expression in embryos is well conserved with vertebrate homologues. Its expression in the tailbud, a posterior growth zone, is consistent with expression seen in other bilateri