The evolution of mating systems in leafroller moths involves differential regulation of a desaturase gene that produces distinct sex pheromones. In the genus Planotortrix, female P. octo predominantly emits (Z)-8-tetradecenyl acetate due to the expression of desat5 in their pheromone glands, whereas female P. excessana produces a blend of (Z)-5- and (Z)-7-tetradecenyl acetate and does not express desat5. In this study, F1 females from interspecific crosses of these species and F1 backcrosses to P. excessana expressed little, if any, desat5 in their pheromone glands, whereas F1 backcrosses to P. octo and F2 crosses displayed a range of expression levels of desat5, consistent with the action of a trans-acting repressor from P. excessana. Females expressing desat5 always produced (Z)-8-tetradecenyl acetate, and the presence of at least one P. octo-like allele of desat5 was required for the expression of desat5, suggesting a cis-regulatory factor from P. octo is necessary. Comparison of 1148bp upstream of the desat5 open reading frame revealed 35 differences, including a 7-bp insertion in P. octo. We argue these data best fit a model of pheromone evolution that involves changes in a trans-acting repressor and a cis-regulatory mutation in an activator binding site within the desat5 promoter.