The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based gene-trap screen in mice designed for the large-scale identification of genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.