Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.