Pheromones are central to the mating systems of a wide range of organisms, and reproductive isolation between closely related species is often achieved by subtle differences in pheromone composition. In insects and moths in particular, the use of structurally similar components in different blend ratios is usually sufficient to impede gene flow between taxa. To date, the genetic changes associated with variation and divergence in pheromone signals remain largely unknown. Using the emerging model system Ostrinia, we show the functional consequences of mutations in the protein-coding region of the pheromone biosynthetic fatty-acyl reductase gene pgFAR. Heterologous expression confirmed that pgFAR orthologs encode enzymes exhibiting different substrate specificities that are the direct consequences of extensive nonsynonymous substitutions. When taking natural ratios of pheromone precursors into account, our data reveal that pgFAR substrate preference provides a good explanation of how species-specific ratios of pheromone components are obtained among Ostrinia species. Moreover, our data indicate that positive selection may have promoted the observed accumulation of nonsynonymous amino acid substitutions. Site-directed mutagenesis experiments substantiate the idea that amino acid polymorphisms underlie subtle or drastic changes in pgFAR substrate preference. Altogether, this study identifies the reduction step as a potential source of variation in pheromone signals in the moth genus Ostrinia and suggests that selection acting on particular mutations provides a mechanism allowing pheromone reductases to evolve new functional properties that may contribute to variation in the composition of pheromone signals.