Pax6 is a key transcriptional regulator in eye, olfactory system, forebrain, pituitary cerebellum, spinal cord and pancreas development. Alternative splicing, promoter usage and multiple enhancers regulate the complex Pax6 spatio-temporal expression pattern. Chromosomal rearrangements which abolish PAX6 gene expression have been characterised downstream of the coding region. Through evolutionary sequence comparison and transgenic reporter studies, we have identified a new Pax6 3' cis-regulatory region. This region, C1170 Box 123, contains three distinct modules of human-mouse sequence conservation, while only Box 1 is conserved to Fugu. Both the human and the orthologous Fugu sequence direct similar reporter gene expression in the developing pretectum, neural retina and olfactory region, indicating evolutionary conservation of Pax6 regulatory mechanisms despite the low level of overall sequence conservation.