Whole genome duplication events are thought to have substantially contributed to organismal complexity, largely via divergent transcriptional regulation. Members of the vertebrate PAX2, PAX5 and PAX8 gene subfamily derived from an ancient class of paired box genes and arose from such whole genome duplication events. These genes are critical in establishing the midbrain-hindbrain boundary, specifying interneuron populations and for eye, ear and kidney development. Also PAX2 has adopted a unique role in pancreas development, whilst PAX5 is essential for early B-cell differentiation. The contribution of PAX258 genes to their collective role has diverged across paralogues and the animal lineages, resulting in a complex wealth of literature. It is now timely to provide a comprehensive comparative overview of these genes and their ancient and divergent roles. We also discuss their fundamental place within gene regulatory networks and the likely influence of cis-regulatory elements over their differential roles during early animal development. Developmental Dynamics 238:2951–2974, 2009. © 2009 Wiley-Liss, Inc.