Butterfly eyespot colour patterns are a key example of how a novel trait can appear in association with the co-option of developmental patterning genes. Little is known, however, about how, or even whether, co-opted genes function in eyespot development. Here we use CRISPR/Cas9 genome editing to determine the roles of two co-opted transcription factors that are expressed during early eyespot determination. We found that deletions in a single gene, spalt, are sufficient to reduce or completely delete eyespot colour patterns, thus demonstrating a positive regulatory role for this gene in eyespot determination. Conversely, and contrary to previous predictions, deletions in Distal-less (Dll) result in an increase in the size and number of eyespots, illustrating a repressive role for this gene in eyespot development. Altogether our results show that the presence, absence and shape of butterfly eyespots can be controlled by the activity of two co-opted transcription factors.