show Abstracthide AbstractThe genetic changes underlying novel morphological structures remain poorly understood. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 141 individuals across 17 species identified narrow regions associated with two distinct elements of this pattern which have distinct evolutionary histories. These elements arose from within two Heliconius clades that diverged over 3 million years ago, showing that novel combinations of phenotypic traits can arise through recombination between divergent lineages. We hypothesize that these modules represent distinct cis-regulatory loci that regulate the transcription factor optix. An expression microarray and immunohistochemistry identified the transcription factor Homothorax which co-localises with Optix in the proximal forewing. Sequence analysis and mobility shift assays show acquisition of Hth binding sites in butterflies with red patches on the base of the forewing, demonstrating that this phenotype arose through evolution of a novel regulatory link between a colour-patterning factor (Optix) and a conserved wing pattern factor (Homothorax).