Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult.The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.