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Where their geographic and host ranges overlap, sibling species of tree-killing bark beetles may simultaneously attack and reproduce on the same hosts. However, sustainability of these potentially mutually beneficial associations demands effective prezygotic reproductive isolation mechanisms between the interacting species. The pine bark beetle, Dendroctonus frontalis Zimmermann, is syntopic in the Central American region with a recently described sibling species, Dendroctonus mesoamericanus Armendáriz-Toledano and Sullivan, but mechanisms for their reproductive isolation are uncertain. We investigated whether semiochemicals mediate species discrimination by mate-seeking males of both species. In olfactometer bioassays, walking males of both species strongly preferred odors from gallery entrances of conspecific females. Coupled gas chromatography-electroantennographic detection and gas chromatography-mass spectrometry isolated 16 olfactory stimulants for males in these odors, but only two, ipsdienol and endo-brevicomin (both from D. mesoamericanus females), differed in quantity in female-associated odors between the species. In olfactometer bioassays, with 10, 1, or 0.1 female entrance equivalents of synthetic semiochemicals, the combination of ipsdienol and endo-brevicomin inhibited responses of male D. frontalis and enhanced responses of male D. mesoamericanus to two compounds associated with female entrances of both species (the pheromone component frontalin and host odor α-pinene). We conclude that ipsdienol and endo-brevicomin, pheromone components produced by females of just one of the two species (D. mesoamericanus), mediate interspecific mate discrimination by males of both species and provide an apparently symmetrical reproductive isolation mechanism. Inglés