AbstractChromosomal polymorphism is widespread in the Drosophila genus, with extensive evidence supporting its adaptive character in diverse species. Moreover, inversions are the major contributors to the genus chromosomal evolution. The molecular characterization of a reduced number of polymorphic inversion breakpoints in Drosophila melanogaster and Drosophila subobscura supports that their inversions would have mostly originated through a mechanism that generates duplications —staggered double-strand breaks— and has thus the potential to contribute to their adaptive character. There is also evidence for inversion breakpoint reuse at different time scales. Here, we have characterized the breakpoints of two inversions of D. subobscura —O4 and O8— involved in complex arrangements that are frequent in the warm parts of the species distribution area. The duplications detected at their breakpoints are consistent with their origin through the staggered-break mechanism, which further supports it as the prevalent mechanism in D. subobscura. The comparative analysis of inversions breakpoint regions across the Drosophila genus has revealed several genes affected by multiple disruptions due not only to inversions but also to single-gene transpositions and duplications.