The molecular basis of regionalisation and patterning of the developing brain is an area of current intense interest. Members of the Otx, Pax-2/5/8 and Hox gene families appear to play important roles in these processes in vertebrates, but functional divergence and genetic redundancy arising from gene duplication events obscures our view of the roles played by these genes during the evolution of vertebrate brains. Determination of the ancestral gene copy number in chordates through molecular phylogenetics, accompanied by gene expression analysis in all three chordate subphyla (vertebrates, cephalochordates and urochordates) may distinguish between ancestral and derived expression domains and give clues to the roles played by these genes in chordate ancestors. Application of this comparative approach indicates evolutionary homologous brain regions (fore-/midbrain, isthmus/cerebellum and hindbrain) in chordates and supports homology of the frontal eye of cephalochordates to the paired eyes of vertebrates.