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Analysis of maternally inherited genes is especially helpful in population studies of host-specialized insects, as female dispersal is key to find an adequate host plant to ensure larval survival. Bucculatrix mirnae (Lepidoptera: Bucculatricidae) is a little-known Neotropical micromoth native to the arid environments of northern Chile whose hypermetamorphic larvae are miners and skeletonizers on leaves of two species of Baccharis (Asteraceae) shrubs. This micromoth has been detected in three isolated locations embracing a narrow geographic range: two from the coastal valleys of the Atacama Desert near sea level and one from the western slopes of the Andes at about 3000 m elevation. As the dispersal of B. mirnae is mostly restricted to the small adult stage, the altitudinal gradient and desert areas among the three localities could be effective barriers, triggering genetic differentiation among populations. Sequences of the DNA barcode fragment of the cytocrome oxidase subunit I mitochondrial gene were analyzed to assess for the first time the patterns of genetic variation of B. mirnae. Fifteen haplotypes, each exclusive to one locality, were found in the 71 specimens analyzed. Genetic divergence (K2P) between haplotypes of different localities was at least 2.0%. A Bayesian analysis with sequences of congeneric species grouped all the B. mirnae haplotypes in a Glade, in which three well-supported locality-specific haplogroups were found. In concordance with this pattern, an analysis of molecular variance showed that the highest genetic variation was found among populations. Furthermore, all the population pairwise comparisons (F-ST) were significant. These results suggest that female migration between isolated populations of B. mirnae is absent. This pattern must be considered in the current scenario of habitat destruction and modification in the arid environments of northern Chile.