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BACKGROUND: The sugarcane borer Diatraea saccharalis (Lepidoptera) is a key pest on sugarcane and other grasses in the Americas. Biological control as well as insecticide treatments are used for pest management, but economic losses are still significant. The use of female sex pheromones for mating disruption or mass trapping in pest management could be established for this species, provided that economical production of pheromone is available. RESULTS: Combining in vivo labelling studies, differential expression analysis of transcriptome data and functional characterisation of insect genes in a yeast expression system, we reveal the biosynthetic pathway and identify the desaturase and reductase enzymes involved in the biosynthesis of the main pheromone component (9Z,11E)‐hexadecadienal, and minor components hexadecanal, (9Z)‐hexadecenal and (11Z)‐hexadecenal. We next demonstrate heterologous production of the corresponding alcohols of the pheromone components, by expressing multiple steps of the biosynthetic pathway in yeast. CONCLUSION: Elucidation of the genetic basis of sex pheromone biosynthesis in D. saccharalis, and heterologous expression in yeast, paves the way for biotechnological production of the pheromone compounds needed for pheromone‐based pest management of this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]